WO2025003669A1 - Procédé, kit et système de synthèse de polynucléotides - Google Patents

Procédé, kit et système de synthèse de polynucléotides Download PDF

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WO2025003669A1
WO2025003669A1 PCT/GB2024/051643 GB2024051643W WO2025003669A1 WO 2025003669 A1 WO2025003669 A1 WO 2025003669A1 GB 2024051643 W GB2024051643 W GB 2024051643W WO 2025003669 A1 WO2025003669 A1 WO 2025003669A1
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polynucleotide
strand
acceptor
terminal end
donor
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Tarun Stephen CHAPMAN
David Edward Clarke
James Anthony Clarke
Imogen Helen COOK
Carolin Anne MÜLLER
Ryosuke OGAKI
James Philip WHITE
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Oxford Nanopore Technologies PLC
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Oxford Nanopore Technologies PLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence.
  • the invention also relates to methods for the assembly of synthetic polynucleotides following synthesis.
  • Phosphoramidite chemistry is a synthetic approach involving the assembly of monomers of chemically activated T, C, A or G into oligonucleotides of approximately 100/150 bases in length via a stepwise process.
  • the chemical reaction steps are highly sensitive and the conditions alternate between fully anhydrous (complete absence of water), aqueous oxidative and acidic conditions (Roy and Caruthers, Molecules, 2013, 18, 14268-14284). If the reagents from the previous reaction step have not been completely removed this will be detrimental to future steps of synthesis. Accordingly, this synthesis method is limited to the production of polynucleotides of length of approximately 100 nucleotides.
  • the Polymerase Synthetic approach uses a polymerase to synthesise a complementary strand to a DNA template using T, C, A and G triphosphates.
  • the reaction conditions are aqueous and mild and this approach can be used to synthesise DNA polynucleotides which are many thousands of bases in length.
  • the main disadvantage of this method is that single- and double-stranded DNA cannot be synthesised de novo by this method, it requires a DNA template from which a copy is made, thus limiting its utility (Kosuri and Church, Nature Methods, 2014, 11, 499-507).
  • TdT terminal deoxynucleotidyl transferase
  • This enzyme can be used to extend a single-stranded oligonucleotide in a 5 ’ to 3 ’ direction in a controlled manner.
  • the synthesised singlestranded oligonucleotide can subsequently be converted to a double-stranded molecule using the synthesised single-stranded oligonucleotide as a template.
  • the inventors have developed new methodologies by which single- and doublestranded polynucleotide molecules can be synthesised de novo in a stepwise manner without the need to copy a pre-existing template molecule. Such methods also avoid the extreme conditions associated with phosphoramidite chemistry techniques and in contrast are carried out under mild, aqueous conditions around neutral pH.
  • the invention provides in vitro methods of synthesising a double-stranded polynucleotide having a predefined sequence.
  • the invention is further defined in the section below.
  • An in vitro method of synthesising a double-stranded polynucleotide having a predefined sequence comprising performing cycles of synthesis, wherein each cycle comprises:
  • A providing an acceptor polynucleotide which is blunt-ended and double-stranded
  • B providing a blunt-ended donor polynucleotide having first and second strands, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site
  • step (A) comprises: providing an acceptor polynucleotide comprising first and second polynucleotide strands and first and second terminal ends, wherein the first terminal end is ligatable and blunt-ended;
  • step (B) comprises: providing a donor polynucleotide comprising:
  • first and second terminal ends wherein the first terminal end is ligatable, blunt-ended and comprises a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence, the terminal nucleotide pair being the first pair of the payload;
  • step (C) comprises: ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide; and step (D) comprises: cleaving the ligated polynucleotide at the cleavage site, separating the cleaved donor polynucleotide from the acceptor polynucleotide, generating a blunt end at the cleaved first terminal end of the acceptor polynucleotide, thereby extending the cleaved first terminal end of the acceptor polynucleotide with the polynucleotide payload and generating a new blunt- ended ligatable first terminal end of the acceptor polynucleotide for ligation in the next cycle.
  • (i) comprises a 5’ phosphate group
  • step (C) comprises: (i) joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end; wherein the first strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and (ii) joining the first strands of the donor and acceptor polynucleotides at their first terminal ends; and following step (D) the 5’ phosphate group joined to the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is removed, preferably by the action of an enzyme having phosphatase activity.
  • step C(ii) comprises adding a phosphate group to the first strand of the acceptor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide with the first strand of the acceptor polynucleotide.
  • PNK polynucleotide kinase
  • step (C) comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end; wherein the first strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; following step (C) and before step (D) the method further comprises performing an incorporation step to extend the first strand of the donor polynucleotide at its first terminal end at the nick site, the step comprising synthesising new nucleotides in the first strand of the acceptor polynucleotide using the nucleotides of the second strand as templates, preferably by the action
  • step (C) comprises: (i) joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end; wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and (ii) joining the second strands of the donor and acceptor polynucleotides at their first terminal ends.
  • step C(ii) comprises adding a phosphate group to the second strand of the donor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide with the second strand of the acceptor polynucleotide.
  • PNK polynucleotide kinase
  • step (C) comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end; wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and before step (D) the method further comprises breaking the bonds between nucleotides of the first and second strands of the donor polynucleotide, thereby generating a single stranded donor polynu
  • both polynucleotide strands of the second terminal end of the donor polynucleotide of step (B) are connected together by a polynucleotide hairpin loop which encodes one strand of the cleavage site, and wherein the step of synthesising a new second strand in the donor polynucleotide before step (D) comprises using the nucleotides of the hairpin as templates to generate a complete double stranded cleavage site.
  • step (C) comprises: joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end, and wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and wherein step (D) comprises:
  • step (D) the method further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide and performing an incorporation step comprising extending the second strand of the acceptor polynucleotide at the nick site with new payload
  • step (C) comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end; wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; following step (C) and before step (D) the method further comprises performing a first incorporation step to extend the second strand of the acceptor polynucleotide from the nick site, the step comprising synthesising new nucleotides in the second strand using the nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesis
  • step (D) comprises: cleaving both strands of the ligated polynucleotide to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload
  • step (D) comprises:
  • step (D) comprises:
  • an incorporation step comprising extending the second strand of the cleaved acceptor polynucleotide with new payload nucleotides using the payload nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby reforming the payload nucleotide pairs in the cleaved polynucleotide and thereby forming a ligatable blunt end at the first end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are incorporated and retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload.
  • step (D) comprises cleaving the sugar-phosphate backbone of the strand.
  • cleavage site in the donor polynucleotide is adjacent to the polynucleotide payload and comprises a recognition sequence for a type IIS restriction enzyme, preferably the wherein the cleavage site is an Mlyl cleavage site.
  • cleavage comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.
  • the cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide, wherein following cleavage the terminal nucleotide in the first strand of the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload, optionally wherein the universal nucleotide is inosine.
  • each cleavage step comprises a two-step cleavage process wherein each cleavage step comprises a first step comprising removing the universal nucleotide to form an abasic site, and a second step comprising cleaving the first strand of the donor polynucleotide at the abasic site.
  • nucleotide-excising enzyme is a 3 -methyladenine DNA glycosylase enzyme.
  • nucleotide-excising enzyme is: i. human alkyladenine DNA glycosylase (hAAG); or ii. uracil DNA glycosylase (UDG).
  • each cleavage step comprises a one step cleavage process comprising removing the universal nucleotide with a cleavage enzyme wherein the enzyme is
  • cleavage step comprises cleaving the first strand of the donor polynucleotide with an enzyme.
  • cleavage site is defined by a uracil nucleotide positioned in the first strand of the donor polynucleotide, wherein cleavage is performed by an enzyme having uracil DNA glycosylase activity and DNA glycosylase- lyase activity e.g. Endonuclease VIII activity, and wherein following cleavage the terminal nucleotide of first strand at the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • an enzyme having uracil DNA glycosylase activity and DNA glycosylase- lyase activity e.g. Endonuclease VIII activity
  • a method according to any one of the preceding aspects wherein ligation is performed by the action of an enzyme having nucleotide ligase activity.
  • the enzyme is human DNA ligase III, T3 DNA ligase, T4 DNA ligase, optionally T4 DNA ligase which has improved thermal stability compared to wild-type T4 DNA ligase, preferably wherein the enzyme is a T3 DNA ligase or a T4 DNA ligase which has improved salt tolerance compared to wildtype T4 DNA ligase.
  • the terminal nucleotide of the first and/or second strands of the second terminal end of the donor polynucleotide comprises a blocking group
  • both polynucleotide strands of the second terminal end of the donor polynucleotide are connected together, preferably by a polynucleotide hairpin loop.
  • the 3 ’ terminal nucleotide of the second terminal end of the donor polynucleotide comprises a phosphate group, 2 '-3 '-dideoxycytidine, inverted deoxythymidine or a spacer, optionally an ethylene glycol based spacer e.g. hexanediol; and/or
  • the 5 ’ terminal nucleotide of the second terminal end of the donor polynucleotide comprises 2 '-3 '-dideoxycytidine, inverted deoxythymidine or a spacer, optionally an ethylene glycol based spacer e.g. hexanediol; optionally wherein the 3 ’ terminal nucleotide of the second terminal end of the donor polynucleotide comprises a phosphate group and the 5 ’ terminal nucleotide has no blocking group.
  • a method according to aspect 44 wherein the method is performed by a method according to any one of aspects 17 to 43.
  • 47 A method according to any one of aspects 1 to 3, 8, 9 and 12 to 16 wherein the second terminal end of the donor polynucleotide comprises a second polynucleotide payload, wherein:
  • the payload nucleotide sequence of the first strand at the second terminal end of the donor polynucleotide in the 5 ’ to 3 ’ direction is the same as the payload nucleotide sequence of the second strand at the first terminal end of the donor polynucleotide in the 5 ’ to 3 ’ direction;
  • the payload nucleotide sequence of the second strand at the second terminal end of the donor polynucleotide in the 3 ’ to 5 ’ direction is the same as the payload nucleotide sequence of the first strand at the first terminal end of the donor polynucleotide in the 3 ’ to 5 ’ direction;
  • the second terminal end of the donor polynucleotide comprises a 3 ’ hydroxyl group and lacks a 5’ phosphate group.
  • a method according to aspect 47 wherein the method is performed by a method according to any one of aspects 17 to 43.
  • step (D) further comprises:
  • polynucleotide payload consists of two or more, or three or more consecutive pairs of nucleotides of the predefined sequence.
  • polynucleotide payload consists of four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive pairs of nucleotides of the predefined sequence.
  • step (D) of aspect 1 comprises performing a single cleavage step comprising cleaving the ligated polynucleotide and generating a cleaved blunt end, thereby extending the acceptor polynucleotide with the polynucleotide payload at the cleaved end, and generating a new blunt-ended double-stranded acceptor polynucleotide for ligation and extension in the next cycle.
  • each cycle does not involve a step of incorporation of a polynucleotide having a reversible terminator group and an additional step of deprotection to remove the reversible terminator group.
  • the first and second strands of the acceptor polynucleotide at the second terminal end are each tethered to a surface; or (ii) the first and second strands of the acceptor polynucleotide at the second terminal end are connected together by a polynucleotide hairpin loop and are tethered to a surface; or
  • the surface comprises a polyacrylamide surface, such as about 2% polyacrylamide, preferably wherein the polyacrylamide surface is coupled to a solid support such as glass.
  • a method according to aspect 65 wherein the one or more covalent bonds is formed between a functional group on the surface and a functional group on the acceptor polynucleotide, wherein the functional group on the acceptor polynucleotide is an amine group, a thiol group, a thiophosphate group or a thioamide group.
  • BRAPA N- (5-bromoacetamidylpentyl) acrylamide
  • microfluidic system is an electrowetting system.
  • microfluidic system is an electrowetting-on-dielectric system (EWOD).
  • EWOD electrowetting-on-dielectric system
  • a method according to any one of the preceding aspects wherein following synthesis the strands of the double-stranded polynucleotide having a predefined sequence are separated to provide a single-stranded polynucleotide having a predefined sequence.
  • 72. A method according to any one of the preceding aspects, wherein following synthesis the double-stranded polynucleotide having a predefined sequence, or a region thereof, is amplified, preferably by PCR.
  • a method of assembling a polynucleotide having a predefined sequence comprising performing the method of any one of the preceding aspects to synthesise a first polynucleotide having a predefined sequence and one or more additional polynucleotides having a predefined sequence and joining together the first and one or more additional polynucleotides.
  • a method according to aspect 76 wherein the first polynucleotide and the one or more additional polynucleotides are cleaved by a restriction enzyme at a cleavage site.
  • a method according to aspect 78 wherein the assembly steps comprise providing a first droplet comprising a first synthesised polynucleotide having a predefined sequence and a second droplet comprising an additional one or more synthesised polynucleotides having a predefined sequence, wherein the droplets are brought in contact with each other and wherein the synthesised polynucleotides are joined together thereby assembling a polynucleotide comprising the first and additional one or more polynucleotides.
  • a method according to aspect 80 wherein following delivery of a droplet and prior to the delivery of a next droplet, a washing step is carried out to remove excess reaction reagents.
  • micro fluidic system is an electrowetting system.
  • micro fluidic system is an electrowetting-on-dielectric system (EWOD).
  • EWOD electrowetting-on-dielectric system
  • Figure 1 depicts symbols and terminology used in the schematics of the chemistries of the specific exemplary structures and synthesis methods outlined in Figures 3 to 9.
  • Figure 2
  • Figure 2 depicts a general non-limiting exemplary structure of an acceptor polynucleotide as used in the chemistries of the specific exemplary synthesis methods outlined in Figures 4 to 9.
  • Figure 3 depicts general non-limiting exemplary structures of donor polynucleotides as used in the chemistries of the specific exemplary synthesis methods outlined in Figures 4 to 9.
  • Figure 4 depicts the chemistry and methology of the specific exemplary synthesis method version 1.
  • Figure 5 depicts the chemistry and methology of the specific exemplary synthesis method version 2.
  • Figure 6 depicts the chemistry and methology of the specific exemplary synthesis method version 3.
  • Figure 7 depicts the chemistry and methology of the specific exemplary synthesis method version 4.
  • Figure 8 depicts the chemistry and methology of the specific exemplary synthesis method version 4.
  • Figure 8 depicts the chemistry and methology of the specific exemplary synthesis method version 5.
  • Figure 9 depicts the chemistry and methology of the specific exemplary synthesis method version 6.
  • Figure 10 depicts various cleavage mechanisms in methods where the cleavage site is defined by a universal nucleotide.
  • Figure 11 presents schemes showing examples of surface chemistries for attaching polynucleotides to surfaces.
  • the examples show double-stranded embodiments wherein both strands are connected via a hairpin, but the same chemistries may be used for attaching one or both strands of a double-stranded polynucleotide where the strands are not connected via a hairpin.
  • Figure 12 presents schemes showing examples of surface chemistries for attaching polynucleotides to surfaces.
  • the examples show double-stranded embodiments wherein both strands are connected via a hairpin, but the same chemistries may be used for attaching one or both strands of a double-stranded polynucleotide where the strands are not connected via a hairpin.
  • Figure 13 presents schemes showing examples of surface chemistries for attaching polynucleotides to surfaces.
  • the examples show double-stranded embodiments wherein both strands are connected via a hairpin, but the same chemistries may be used for attaching one or both strands of a double-stranded polynucleotide where the strands are not connected via a hairpin.
  • Figure 13 presents schemes showing examples of surface chemistries for attaching polynucleotides to surfaces.
  • the examples show double-stranded embodiments wherein both strands are connected via a hairpin, but
  • Figure 13 depicts and explains structural features relating to non-limiting exemplary Examples 1 to 3.
  • Figure 14 depicts the experimental results relating to methods described in Example 3.
  • the present invention provides methods for the de novo synthesis of polynucleotide molecules according to a predefined nucleotide sequence.
  • Synthesised polynucleotides are preferably DNA and are preferably double-stranded polynucleotide molecules.
  • the present inventors have utilised a donor polynucleotide which carries a polynucleotide payload.
  • the donor polynucleotide is ligated to an acceptor polynucleotide to form a ligated polynucleotide.
  • the ligated polynucleotide is then cleaved. Cleavage is structured such that the polynucleotide payload, which was originally part of the donor polynucleotide, is retained as part of the acceptor polynucleotide.
  • the invention provides an in vitro method of synthesising a double-stranded polynucleotide molecule having a predefined sequence, the method comprising performing cycles of synthesis, wherein each cycle comprises:
  • the methods are performed to synthesise a DNA polynucleotide having a predefined sequence.
  • the methods may also be performed to synthesise an RNA polynucleotide having a predefined sequence.
  • the methods may be performed to synthesise a double-stranded polynucleotide having a predefined sequence.
  • the methods may be performed to synthesise a double-stranded polynucleotide having a predefined sequence wherein one strand is a DNA strand and the other strand is an RNA strand.
  • the two strands of a double-stranded polynucleotide synthesised in accordance with the methods of the invention may be separated to form a single-stranded polynucleotide having a predefined sequence.
  • the methods of the invention may therefore be performed to form a single-stranded DNA polynucleotide having a predefined sequence, or a single-stranded RNA polynucleotide having a predefined sequence.
  • the invention is not limited to synthesising exclusively DNA or RNA molecules, and other forms of polynucleotide may be synthesised as discussed further herein.
  • the invention provides advantages compared with existing synthesis methods. For example, all reaction steps may be performed in aqueous conditions at mild pH, extensive protection and deprotection procedures are not required. Furthermore, synthesis is not dependent upon the copying of a pre-existing template strand comprising the predefined nucleotide sequence. A further advantage arises from the feature requiring the single-stranded ligation, in step (C), of only the first strands of the acceptor and donor polynucleotides, or the single-stranded ligation of only the second strands of the acceptor and donor polynucleotides.
  • Such single-stranded ligation steps are therefore deliberately directed to ligate only the first strands and not the second strands, or only the second strands and not the first strands. Such single -stranded ligation steps are therefore not random or indiscriminate as between the pairs of first and second strands.
  • This allows a donor polynucleotide to be provided in step (B) such that a terminal end which is ligatable to the first terminal end of an acceptor polynucleotide in step (C) may at the same time be not self-ligatable. Avoiding selfligation of donor polynucleotides minimises reagent loss and increases the efficiency of synthesis reactions.
  • steps (A) and (B) provides flexibility in terms of the number of donor polynucleotide species required to deliver payloads for the generation of a double-stranded polynucleotide having a given predefined sequence.
  • the invention provides an in vitro method for synthesising a double-stranded polynucleotide having a predefined sequence.
  • Synthesis is carried out under conditions suitable for hybridization of nucleotides within double-stranded polynucleotides.
  • Polynucleotides are typically contacted with reagents under conditions which permit the hybridization of nucleotides to complementary nucleotides.
  • Conditions that permit hybridization are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscience, New York (1995)).
  • Ligation of polynucleotides can be carried out under suitable conditions, for example using a ligase (e.g., T4 DNA ligase) at a temperature that is compatible with the enzyme (e.g., room temperature) in the presence of a suitable buffered solution.
  • a ligase e.g., T4 DNA ligase
  • the buffered solution can comprise 4.4 mM Tris-HCl, 7mM MgCh, 0.7mM dithiothreitol, 0.7mM ATP, 5% polyethylene glycol (PEG6000).
  • Cleavage of polynucleotides can be carried out under suitable conditions, for example using a polynucleotide cleaving enzyme (e.g., endonuclease) at a temperature that is compatible with the enzyme (e.g., 37°C) in the presence of a suitable buffered solution.
  • a polynucleotide cleaving enzyme e.g., endonuclease
  • the buffered solution can comprise 5 mM potassium acetate, 2 mM Tris-acetate, 1 mM magnesium acetate and 0.1 mM DTT.
  • incorporation of nucleotides into polynucleotides can be carried out under suitable conditions, for example using a polymerase (e.g. , Therminator IX polymerase) or a terminal deoxynucleotidyl transferase (TdT) enzyme or functional variant thereof to incoprorate nucleotides at a suitable temperature (e.g., ⁇ 65°C) in the presence of a suitable buffered solution.
  • the buffered solution can comprise 2 mM Tris-HCl, 1 mM (NH 4 ) 2 SO4, 1 mM KC1, 0.2 mM MgSO 4 and 0.01% Triton® X-100.
  • the methods of the invention involve synthesising a double-stranded polynucleotide molecule having a predefined sequence.
  • predefined sequence it is meant that the nucleotide sequence of the polynucleotide molecule is determined by the user before the method is performed. The method is therefore performed in a manner that results in the final de novo synthesised polynucleotide molecule having the nucleotide sequence that was determined by the user before synthesis.
  • the methods do not require the “copying”, via complementary Watson-Crick base-pairing, of a “template” polynucleotide strand that existed before the method was performed.
  • Each one of the specific exemplary methods of the present invention involves the use of an acceptor polynucleotide.
  • Acceptor polynucleotides are described extensively with respect to the specific chemistry methods of the invention set out below and are depicted visually in the corresponding figures.
  • an acceptor polynucleotide acts to accept a double-stranded polynucleotide payload consisting of nucleotides of the predefined sequence.
  • the polynucleotide payload is further described herein. Successive cycles of synthesis lead to the stepwise addition of multiple polynucleotide payloads leading to the generation of the polynucleotide having a predefined sequence. Accordingly the acceptor polynucleotide acts as a scaffold on which the polynucleotide having a predefined sequence is synthesised.
  • the acceptor polynucleotide is blunt-ended and double-stranded.
  • the acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the acceptor polynucleotide is ligatable and blunt- ended.
  • ligatable it is meant that it is capable of being ligated to the first terminal end of a donor polynucleotide, as described and defined further herein.
  • a “ligatable” terminal end of an acceptor polynucleotide may be interpreted as, or explicitly referred to as, “donor ligatable” or “donor polynucleotide ligatable”.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide may or may not comprise a 5’ phosphate group. Whether or not a 5 ’ phosphate group is present will depend upon the specific chemistry method employed.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end does not comprise a 5’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end may or may not comprise a 3’ hydroxyl group. Whether or not a 3 ’ hydroxyl group is present will depend upon the specific chemistry method employed at the preference of the user.
  • the second terminal end of the acceptor polynucleotide is preferably non-ligatable.
  • non-ligatable it is meant that it is not capable of being ligated to another nucleic acid molecule, including to the first terminal end of a donor polynucleotide as described and defined further herein.
  • the second terminal end of the acceptor polynucleotide is preferably tethered to a surface, such as depicted in Figure 2.
  • a surface may be any suitable surface as described and defined elsewhere herein.
  • the second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered, such as depicted in Figure 2.
  • the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface.
  • first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.
  • the acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesise. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include pairs of nucleotides of the predefined sequence at the first terminal end, for example where the acceptor polynucleotide is the product of a previous cycle of synthesis.
  • the first and second strands of the acceptor polynucleotide may comprise nucleotides, nucleotide analogues/derivatives and/or non-nucleotides.
  • acceptor polynucleotide comprising first and second strands which will be suitable to facilitate ligation, as described further herein, and which are capable of priming new polynucleotide synthesis as described further herein if desired.
  • mismatches between strands should be avoided, GC- and AT-rich regions should be avoided, and in addition regions of secondary structure such as hairpins or bulges which might interfere with ligation and/or other extension should be avoided.
  • the length of the first and second strands of the acceptor polynucleotide can be chosen by the skilled person depending on preference and the ligase enzyme to be used.
  • the first strand is hybridized to the corresponding region of the second strand. It is not essential that the entirety of the first strand is hybridized to the corresponding region of the second strand, provided that first and second strands are suitable for ligation as described herein, or capable of priming new polynucleotide synthesis as described further herein if desired. Thus, mismatches between the first strand and the corresponding region of the second strand can be tolerated to a degree.
  • the region of sequence of the first and second strands at the end of the acceptor polynucleotide to be extended should comprise nucleobases which are complementary to corresponding nucleobases in the opposite strand.
  • the first strand may be connected to the corresponding region of the second strand at the end of the acceptor polynucleotide which is not to be extended, i.e. the second terminal end, e.g. via a hairpin.
  • acceptor polynucleotide A non-limiting exemplary embodiment of an acceptor polynucleotide is provided in the Examples below.
  • Each one of the specific exemplary methods of the present invention involves the use of a donor polynucleotide.
  • Donor polynucleotides are described extensively with respect to the specific chemistry methods of the invention set out below and nonlimiting exemplary donor polynucleotides are depicted visually in the corresponding Figures.
  • a donor polynucleotide acts to donate a double-stranded polynucleotide payload consisting of nucletotides of the predefined sequence.
  • the polynucleotide payload is further described herein.
  • the donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • a terminal end of a donor polynucleotide may be “ligatable”.
  • ligatable it is meant that it is capable of being ligated to the first terminal end of an acceptor polynucleotide, as described and defined further herein.
  • a “ligatable” terminal end of a donor polynucleotide may interpreted as, or explicitly referred to as, “acceptor ligatable” or “acceptor polynucleotide ligatable”.
  • acceptor ligatable or “acceptor polynucleotide ligatable”.
  • a skilled person will readily appreciate how a terminal end of a donor polynucleotide may be structured so as to be capable of being ligated to the first terminal end of an acceptor polynucleotide. Further details are provided with reference to the specific method versions described further herein.
  • a terminal end of a donor polynucleotide may be “ligatable” and at the same time it may be structured so that it cannot be ligated to another donor polynucleotide, e.g. another donor polynucleotide of the same structure (excepting for variations in the payload sequence), in a self-ligation reaction. Accordingly, a terminal end of a donor polynucleotide may be “non-self-ligatable”.
  • a skilled person will readily appreciate how a terminal end of a donor polynucleotide may be structured so as to be incapable of being ligated to another donor polynucleotide. Further details are provided with reference to the specific method versions described further herein.
  • a terminal end of a donor polynucleotide may be “non-ligatable”.
  • a terminal end of a donor polynucleotide as “non-ligatable” it is meant that it is not capable of being ligated to the first terminal end of an acceptor polynucleotide and it is also not capable of being ligated to another donor polynucleotide, i.e. it is also “non- self-ligatable”.
  • a skilled person will readily appreciate how a terminal end of a donor polynucleotide may be structured so as to be incapable of being ligated to the first terminal end of an acceptor polynucleotide and to another donor polynucleotide.
  • the second terminal end of a donor polynucleotide may be non-ligatable.
  • the second terminal end of a donor polynucleotide may be non-ligatable, such as in the asymmetrical donor polynucleotide depicted in Figure 3 A.
  • the second terminal end of a donor polynucleotide may be ligatable, such as in the asymmetrical donor polynucleotide depicted in Figure 3B.
  • step (B) may comprise providing a donor polynucleotide having first and second strands, and first and second terminal ends, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site, wherein the first terminal end is blunt-ended and ligatable.
  • step (B) may comprise providing a donor polynucleotide having first and second strands, and first and second terminal ends, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site, wherein the first terminal end is blunt-ended, ligatable and non-self-ligatable.
  • step (B) may comprise providing a donor polynucleotide having first and second strands, and first and second terminal ends, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site, wherein the first terminal end is blunt-ended, ligatable and non-self-ligatable and the second terminal end is either: (i) blunt-ended, ligatable and non-self-ligatable; or (ii) non- ligatable.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the donor polynucleotide may or may not comprise a phosphate group. Whether or not a phosphate group is present will depend upon the specific chemistry method employed.
  • the terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide may or may not comprise a phosphate group. Whether or not a phosphate group is present will depend upon the specific chemistry method employed.
  • the terminal nucleotide of the first or second second strand at the ligatable first terminal end of the donor polynucleotide may or may not comprise a hydroxyl group. Whether or not a hydroxyl group is present will depend upon the specific chemistry method employed at the preference of the user.
  • the second terminal end of the donor polynucleotide is preferably not tethered to a surface, such as the donor polynucleotides depicted in Figure 3.
  • the terminal nucleotides of the first and/or second strands at the second terminal end may comprise a blocking group.
  • a blocking group is any blocking group defined elsewhere herein.
  • a blocking group(s) renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be provided without a 5’ phosphate group.
  • the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.
  • the donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence.
  • the polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation.
  • the terminal nucleotide of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload.
  • the penultimate nucleotide of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.
  • a payload may comprise only a single nucleotide pair of the predefined sequence, in this case the payload may be referred to as a nucleotide payload instead of a polynucleotide payload.
  • a polynucleotide payload may consist of two or more, or three or more consecutive pairs of nucleotides of the predefined sequence.
  • a polynucleotide payload may consist of four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive pairs of nucleotides of the predefined sequence.
  • the donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload.
  • the cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide.
  • the exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below.
  • first and second strands of the donor polynucleotide are suitable to facilitate ligation and cleavage as described further herein, e.g. in relation to specific non-limiting exemplary method versions.
  • the first and second strands of the donor polynucleotide may comprise nucleotides, nucleotide analogues/derivatives and/or non-nucleotides.
  • the polynucleotide payload sequence may be any sequence desired by the user. It is not a requirement that nucleotide pairs are formed of pairs having perfect Watson- Crick complementarity. Mismatches between nucleotides at the same position in the first and second strands can be tolerated. GC- and AT -rich regions may be included if desired. Regions of secondary structure such as hairpins or bulges which might interfere with ligation should however be avoided.
  • the length of the first and second strands of the donor polynucleotide can be chosen by the user depending on preference and the ligase enzyme to be used.
  • the first strand is hybridized to the corresponding region of the second strand. It is not essential that the entirety of the first strand is hybridized to the corresponding region of the second strand, provided that first and second strands are suitable for ligation as described herein. Thus, mismatches between the first strand and the corresponding region of the second strand can be tolerated to a degree.
  • Donor polynucleotides may comprise blocking groups.
  • a blocking group with respect to a donor polynucleotides functions to prevent two or more donor polynucleotides ligating together in a self-ligation reaction. If this were to occur it could reduce the efficiency of and interfere with the ligation step.
  • a blocking group is typically attached to or configured in one or both strands of the second terminal end of the donor polynucleotide.
  • a blocking group may be attached to the 5 ’ terminal nucleotide of the second terminal end of the donor polynucleotide and may be 2 '-3 '-dideoxycytidine, inverted deoxythymidine or a spacer, such as an ethylene glycol based spacer e.g. hexanediol.
  • the 5 ’ terminal nucleotide of the second terminal end of the donor polynucleotide may alternatively be dephosphorylated as a means to block self-ligation.
  • a blocking group may preferably be attached to the 3 ’ terminal nucleotide of the second terminal end of the donor polynucleotide and may be a phosphate group, 2'-3'- dideoxycytidine, inverted deoxythymidine or a spacer, such as an ethylene glycol based spacer e.g. hexanediol.
  • the 5’ terminal nucleotide may have no blocking group.
  • the donor polynucleotide may be a symmetrical donor polynucleotide as described further herein.
  • a symmetrical donor polynucleotide may comprise a blocking group attached to the 3 ’ terminal nucleotide of the first terminal end of the donor polynucleotide and a blocking group attached to the 3 ’ terminal nucleotide of the second terminal end of the donor polynucleotide.
  • the blocking group in each case may be the same, such as a phosphate group.
  • a blocking group may be a hairpin loop, or other type of secondary structure, which connects the first and second strands of the donor polynucleotide at the second terminal end of the donor polynucleotide.
  • the cleavage step functions to cleave the ligated polynucleotide such that the one or more nucleotide pairs of the predefined sequence comprising the polynucleotide payload that was previously part of the donor polynucleotide becomes incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide.
  • the cleavage step functions to separate the polynucleotide payload from the remainder of the donor polynucleotide.
  • the remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.
  • the cleavage step may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide. Cleavage is performed so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.
  • Cleavage is performed such that following cleavage the first terminal end of the acceptor polynucleotide comprising the polynucleotide payload is ligatable.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a phosphate group will depend upon the specific chemisty of the method employed at the preference of the user.
  • the terminal nucleotide of the second strand at the ligatable first terminal end of the acceptor polynucleotide may or may not comprise a hydroxyl group. Wherther or not the terminal nucleotide of the second strand at the ligatable first terminal end comprises a hydroxyl group will depend upon the specific chemisty of the method employed at the preference of the user. Specific methods are described further herein.
  • the cleavage step can be performed by any suitable means for creating the blunt-ended structure described above.
  • the specific type of cleavage will depend upon the specific chemisty of the method employed at the preference of the user.
  • Cleavage may comprise a double-stranded cleavage reaction wherein both the first and second strands are cleaved.
  • both the first and second strands are cleaved at the same positions in a symmetrical cleavage reaction.
  • each strand cleavage is performed at the position between the final nucleotide in the polynucleotide payload and the next nucleotide in the strand in the direction proximal to the second terminal end of the donor polynucleotide/distal to the second terminal end of the acceptor polynucleotide.
  • the terminal nucleotides of the first and second strands at the first terminal end of the acceptor polynucleotide are the final pair of nucleotides of the polynucleotide payload.
  • nick site is introduced into one strand.
  • a nick site is a single-stranded break or gap between nucleotides of a nucleotide strand.
  • a symmetrical cleavage reaction is performed in a method where a nick site is introduced into one strand, following cleavage the original nucleotides of the polynucleotide payload of the nicked strand may remain attached only via interaction (e.g. hydrogen bonding) with the original nucleotides of the polynucleotide payload of the opposite strand.
  • These original nucleotides of the polynucleotide payload of the nicked strand may be removed and replaced by incorporation of new nucleotides of the polynucleotide payload by extension from the nick site.
  • the original nucleotides of the polynucleotide payload of the nicked strand may be separated from the opposite strand:
  • incorporation steps may be performed:
  • cleavage may be performed in such a way that the first strand is cleaved at a different relative position compared to the second strand. Such a cleavage step consequently results in an asymmetrical cleavage. This can be achieved in two ways.
  • both the first and second strands are cleaved, and at different relative positions. This generages a staggered/asymmetrical break.
  • a nick site is introduced into one strand only and the opposite strand is cleaved.
  • the opposite strand is cleaved at a different position relative to the nick site. This also generages a staggered/asymmetrical break.
  • the cleavage step may comprise cleaving only the first strand of the ligated polynucleotide where the second strand comprises a nick site.
  • the cleavage step may comprise cleaving only the second strand of the ligated polynucleotide where the first strand comprises a nick site.
  • the first strand is cleaved immediately above the nucleotides of the polynucleotide payload. I.e. cleavage of the first strand is performed at the position between the final nucleotide in the polynucleotide payload and the next nucleotide in the strand in the direction proximal to the second terminal end of the donor polynucleotide/distal to the second terminal end of the acceptor polynucleotide.
  • the second strand is cleaved below the final nucleotide in the polynucleotide payload in the direction proximal to the second terminal end of the acceptor polynucleotide/distal to the second terminal end of the donor polynucleotide. Accordingly, the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide in the first strand, wherein the terminal nucleotide of the first strand of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage. Following cleavage, such a method further comprises separating the remainder of the donor polynucleotide from the acceptor polynucleotide. Accordingly, because the original nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage, they are consequently discarded. These steps generate an overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • nucleotides of the first strand of the polynucleotide payload overhang the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the polynucleotide payload.
  • the methods further comprise performing an incorporation step comprising extending the second strand of the acceptor polynucleotide by incorporating new payload nucleotides using the original payload nucleotides of the first strand in the overhang as templates, thereby re-forming the payload nucleotides in the second strand, thereby re-forming the payload nucleotide pairs in the ligated polynucleotide and thereby forming a ligatable blunt end at the first end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are incorporated at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides
  • cleavage of the first strand of the ligated polynucleotide is performed in such a way that a 5 ’ phosphate group may be retained on the terminal nucleotide of the first strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the first strand.
  • This is achieved as a consequence of any standard cleavage reaction.
  • the 5 ’ phosphate group may be removed in a dephosphorylation step.
  • incorporation is performed in such a way that a 3 ’ hydroxyl group is retained on the terminal nucleotide of the second strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the second strand.
  • a method is performed wherein following cleavage and following incorporation steps, the first terminal end of the cleaved acceptor polynucleotide comprising the polynucleotide payload is ligatable in the method employed and is thus competent to be ligated to a further donor polynucleotide in the next cycle of synthesis.
  • the cleavage step can be performed by any suitable means for creating the cleaved structures described above and herein.
  • cleavage may comprise a double-stranded cleavage wherein both the first and second strands are cleaved. Such a cleavage step is consequently performed as a symmetrical cleavage, so as to form a blunt end at the cleaved first terminal end of the donor polynucleotide.
  • Cleavage may comprise cleaving the sugarphosphate backbone of the first and second strands of the donor polynucleotide molecule.
  • cleavage may be performed by a restriction enzyme.
  • cleavage may be performed by a type IIS restriction enzyme.
  • the type IIS restriction enzyme may be Mlyl.
  • cleavage site in the donor polynucleotide in a manner that allows the required structure described above to be formed following cleavage.
  • cleavage may comprise a single-stranded cleavage wherein only the first strand is cleaved. Cleavage of only the first strand can be performed in view of the nick site introduced previously into the second strand.
  • a single-stranded cleavage may comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.
  • Single-stranded cleavage may thus be performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, such as BspQI.
  • an enzyme having overhang cleavage function preferably a type IIS restriction enzyme, such as BspQI.
  • Single-stranded cleavage may alternatively be performed by the action of an enzyme having nicking cleavage function, preferably a type IIS restriction enzyme, optionally Nt.&/?01.
  • an enzyme having nicking cleavage function preferably a type IIS restriction enzyme, optionally Nt.&/?01.
  • Single-stranded cleavage may alternatively be performed using a method wherein the cleavage site is defined by a uracil nucleotide and cleavage is performed by the combined action of a Uracil DNA glycosylase enzyme and a DNA glycosylase- lyase enzyme such as Endonuclease VIII.
  • the Uracil DNA glycosylase enzyme catalyses the excision of the uracil base, thus forming an abasic (apyrimidinic) site while at the same time leaving the phosphodiester backbone intact.
  • the DNA glycosylase-lyase enzyme activity creates a break in the phosphodiester backbone at the 3 ' and 5 ' sides of the abasic site, thus generating a single-strand break.
  • Single-stranded cleavage may alternatively be performed using a method wherein the cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide, wherein following cleavage the terminal nucleotide in the first strand of the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • the universal nucleotide may be inosine or any other universal nucleotide described herein. Cleavage mechanisms using universal nucleotides are described elsewhere herein.
  • cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide.
  • a nucleotide position in the first strand may be referred to as positon “n”.
  • the final nucleotide in the polynucleotide payload i.e. the final nucleotide of the polynucleotide payload that is most proximal to the second terminal end of the donor polynucleotide, always occupies position n.
  • nucleotide which occupies the next nucleotide position in the first strand in the direction distal to the second terminal end of the donor polynucleotide always occupies position n-1.
  • nucleotide at position n-1 will be the penultimate nucleotide in the polynucleotide payload.
  • nucleotide which occupies the next nucleotide position in the first strand in the direction proximal to the second terminal end of the donor polynucleotide always occupies position n+1. This arrangement of position numbering is depicted visually in Figure 10.
  • a method comprises cleaving the first strand between nucleotide positions n and n+1.
  • a cleavage mechanism is depicted visually in Figure 10 A.
  • nucleotide position n the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n
  • the penultimate nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n-1
  • the universal nucleotide in the first strand occupies nucleotide position n+2.
  • Such a method comprises cleaving the first strand between nucleotide positions n and n+1. Such a cleavage mechanism is depicted visually in Figure 10B.
  • nucleotide position n the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n
  • the penultimate nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n-1
  • the universal nucleotide in the first strand occupies nucleotide position n+2+x, wherein x is a whole number from 1 to 10 or more.
  • Such methods comprises cleaving the first strand between nucleotide positions n and n+1. Such cleavage mechanisms are depicted visually in Figure IOC.
  • the selection of the reagent to perform the cleavage step will depend upon the particular method employed. Configuration of the desired cleavage site and selection of the appropriate cleavage reagent will therefore depend upon the specific chemistry employed in the method, as will readily be apparent by reference to the exemplary methods described herein.
  • DNA cleaving enzymes Some examples of DNA cleaving enzymes that may be used are shown in the Table below.
  • a new acceptor polynucleotide is created.
  • the new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with a polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide.
  • the new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the doublestranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.
  • a depletion step may be performed before the cleavage step. Such a step is optional and not essential.
  • the depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide.
  • Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide.
  • Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.
  • an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.
  • a depletion step may comprise performing a treatment step to remove the 5’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide.
  • Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • an enzyme having phosphatase activity such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step.
  • the unreacted acceptor polynucleotide may act as a substrate for a nuclease enzyme, which can act to render an unreacted acceptor polynucleotide inert by degrading the first strand of the unreacted acceptor polynucleotide. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, an unreacted acceptor polynucleotide can act as a substrate for an enzyme having 5’ to 3’ exonuclease activity, which can degrade the first strand.
  • a depletion step may comprise performing a nuclease treatment step to degrade the first strand of the acceptor polynucleotide.
  • a depletion step may comprise performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity to degrade the first strand of the acceptor polynucleotide.
  • the donor polynucleotide for use in that cycle of synthesis should be provided such that the second terminal end of the donor polynucleotide cannot act as a substrate for an enzyme having 5 ’ to 3 ’ exonuclease activity.
  • nucleotides are desirable to perform an incorporation/ extension reaction to incorporate nucleotides into polynucleotides, for example to fill in an overhanging end to create a blunt-ended polynucleotide.
  • Nucleotides which can be incorporated into synthetic polynucleotides or provided in polynucleotide payloads by any of the methods described herein may be nucleotides, nucleotide analogues and modified nucleotides.
  • Nucleotides may comprise natural nucleobases or non-natural nucleobases.
  • Nucleotides may contain a natural nucleobase, a sugar and a phosphate group. Natural nucleobases comprise adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C). One of the components of the nucleotide may be further modified.
  • Nucleotide analogues are nucleotides that are modified structurally either in the base, sugar or phosphate or combination therein and that are still acceptable to a polymerase enzyme as a substrate for incorporation into an oligonucleotide strand.
  • a non-natural nucleobase may be one which will bond, e.g. hydrogen bond, to some degree to all of the nucleobases in the target polynucleotide.
  • a non-natural nucleobase is preferably one which will bond, e.g. hydrogen bond, to some degree to nucleotides comprising the nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C).
  • a non-natural nucleotide may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA) and an unlocked nucleic acid (UNA), a bridged nucleic acid (BNA) or a morpholino, a phosphorothioate or a methylphosphonate.
  • a non-natural nucleotide may comprise a modified sugar and/or a modified nucleobase.
  • Modified sugars include but are not limited to 2’-( -methylribose sugar.
  • Modified nucleobases include but are not limited to methylated nucleobases.
  • Methylation of nucleobases is a recognised form of epigenetic modification which has the capability of altering the expression of genes and other elements such as microRNAs. Methylation of nucleobases occurs at discrete loci which are predominately dinucleotide consisting of a CpG motif, but may also occur at CHH motifs (where H is A, C, or T). Typically, during methylation a methyl group is added to the fifth carbon of cytosine bases to create methylcytosine. Thus modified nucleobases include but are not limited to 5-methylcytosine.
  • Nucleotides of the predefined sequence may be incorporated opposite partner nucleotides to form a nucleotide pair.
  • a partner nucleotide may be a complementary nucleotide.
  • a complementary nucleotide is a nucleotide which is capable of bonding, e.g. hydrogen bonding, to some degree to the nucleotides of the predefined sequence.
  • a nucleotide of the predefined sequence is positioned opposite a naturally complementary partner nucleobase.
  • adenosine may be incorporated opposite thymine and vice versa.
  • Guanine may be incorprated opposite cytosine and vice versa.
  • a nucleotide of the predefined sequence may be positioned opposite a partner nucleobase to which it will bond, e.g. hydrogen bond, to some degree.
  • a partner nucleotide may be a non-complementary nucleotide.
  • a non-complementary nucleotide is a nucleotide which is not capable of bonding, e.g. hydrogen bonding, to the nucleotide of the predefined sequence.
  • a nucleotide of the predefined sequence may be incorporated opposite a partner nucleotide to form a mismatch, provided that the synthesised polynucleotide overall is double-stranded and wherein the first strand is attached to the second strand by hybridization.
  • first nucleic acid molecule of sequence 5’-ACGA-3’ may form a duplex with a second nucleic acid molecule of sequence 5’-TCGT-3’ wherein the G of the first molecule will be positioned opposite the C of the second molecule and will hydrogen bond therewith.
  • a first nucleic acid molecule of sequence 5’-ATGA-3’ may form a duplex with a second nucleic acid molecule of sequence 5’-TCGT-3’, wherein the T of the first molecule will mismatch with the G of the second molecule but will still be positioned opposite therewith and will act as a partner nucleotide.
  • This principle applies to any nucleotide partner pair relationship disclosed herein, including partner pairs comprising universal nucleotides.
  • Nucleotides and nucleotide analogues may preferably be provided as nucleoside triphosphates.
  • nucleoside triphosphates may be incorporated from 2’- deoxyribonucleoside-5’-(9-triphosphates (dNTPs), e.g. preferably via the action of a DNA polymerase enzyme or e.g. via the action of an enzyme having deoxynucleotidyl terminal transferase activity as described herein.
  • Triphosphates can be substituted by tetraphosphates or pentaphosphates (generally oligophosphate). These oligophosphates can be substituted by other alkyl or acyl groups:
  • a polynucleotide payload may comprise one or more modified nucleotides.
  • modified bases which may be useful in e.g. click chemistry, which may be incorporated include the following:
  • bases bearing fluorophores and quenchers which may be incorporated include the following:
  • a universal nucleotide may be used to define a cleavage site, as described further herein.
  • a universal nucleotide is one wherein the nucleobase will bond, e.g. hydrogen bond, to some degree to the nucleobase of any nucleotide of the predefined sequence.
  • a universal nucleotide is preferably one which will bond, e.g. hydrogen bond, to some degree to nucleotides comprising the nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C).
  • the universal nucleotide preferably comprises one of the following nucleobases: hypoxanthine, 4-nitro indole, 5 -nitro indole, 6-nitro indole, 3 -nitropyrrole, nitro imidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5 -nitro indazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring.
  • the universal nucleotide more preferably comprises one of the following nucleosides: 2'-deoxyinosine, inosine, 7-deaza-2’ -deoxyinosine, 7-deaza- inosine, 2-aza-deoxyinosine, 2-aza-inosine, 4-nitroindole 2’-deoxyribonucleoside, 4- nitroindole ribonucleoside, 5-nitroindole 2’ deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2' deoxyribonucleoside, 6-nitroindole ribonucleoside, 3- nitropyrrole 2' deoxyribonucleoside, 3 -nitropyrrole ribonucleoside, an acyclic sugar analogue of hypoxanthine, nitroimidazole 2’ deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole
  • hypoxanthi ⁇ ne 8-azahypoxanthine 2-azahypoxanthine 8 -aminohypoxanthine 2-oxopurine inosine base analogues
  • Universal nucleotides incorporating cleavable bases may also be used, including photo- and enzymatically-cleavable bases, some examples of which are shown below.
  • Pho tocleavable bases include photo- and enzymatically-cleavable bases, some examples of which are shown below.
  • SMUG1 Human single-strand-selective monofunctional uracil-DNA Glycosylase
  • ROS1 5 -methylcytosine DNA glycosylase
  • a preferred universal nucleotide is 2 ’-deoxyinosine.
  • first and second strands may be separated. One strand may be discarded and the other strand may be copied to provide a copied strand which has a nucleotide sequence which is complementary to the template strand which is copied. It may be desirable to copy both strands, such as in an amplification reaction e.g. PCR, or any alternative method as described further herein. In any such method any suiable enzyme may be provided to copy the template strand, such as a polymerase enzyme.
  • modified nucleotides such as nucleotides having attached reversible terminator groups, as described herein, in which case polymerase enzymes may be chosen based on their ability to incorporate modified nucleotides.
  • the polymerase may be a modified polymerase having an enhanced ability to incorporate a nucleotide comprising a reversible terminator group compared to an unmodified polymerase.
  • the polymerase is more preferably a genetically engineered variant of the native DNA polymerase from Thermococcus species 9°N, preferably species 9°N-7.
  • modified polymerases are Therminator IX DNA polymerase and Therminator X DNA polymerase available from New England BioLabs. This enzyme has an enhanced ability to incorporate 3’-(9-modified dNTPs.
  • Examples of other polymerases that can be used for incorporation of reversible terminator dNTPs in any of the methods of the invention are Deep Vent (exo-), Vent (Exo-), 9°N DNA polymerase, Therminator DNA polymerase, Therminator IX DNA polymerase, Therminator X DNA polymerase, Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase, Sulfolobus DNA polymerase I, and Taq Polymerase.
  • polymerases examples include T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, pol lambda, pol micro or 029 DNA polymerase.
  • a DNA polymerase may be used to copy a template strand comprising DNA. Any suitable DNA polymerase may be used.
  • the DNA polymerase may be for example Bst DNA polymerase full length, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, E. coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reverse transcriptase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, Taq DNA polymerase, T4 DNA polymerase, T7 DNA polymerase and enzymes having reverse transcriptase activity, for example M-MuLV reverse transcriptase.
  • the DNA polymerase may lack 3’ to 5’ exonuclease activity.
  • Any such suitable polymerase enzyme may be used.
  • a DNA polymerase may be, for example, Bst DNA polymerase full length, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3 >5 ’ exo-), M-MuLV reverse transcriptase, Sulfolobus DNA polymerase IV, Taq DNA polymerase.
  • the DNA polymerase may possess strand displacement activity. Any such suitable polymerase enzyme may be used.
  • Such a DNA polymerase may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3 >5 ’ exo-), M-MuLV reverse transcriptase, phi29 DNA polymerase.
  • the DNA polymerase may lack 3’ to 5’ exonuclease activity and may posess strand displacement activity. Any such suitable polymerase enzyme may be used.
  • Such a DNA polymerase may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, E. coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reverse transcriptase.
  • the DNA polymerase may lack 5 ’ to 3 ’ exonuclease activity. Any such suitable polymerase enzyme may be used.
  • Such a DNA polymerase may be, for example,
  • Bst DNA polymerase large fragment Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment, DNA Pol I large (Klenow) fragment (3 >5 ’ exo-), M-MuLV reverse transcriptase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, T4 DNA polymerase, T7 DNA polymerase.
  • the DNA polymerase may lack both 3’ to 5’ and 5 ’ to 3 ’ exonuclease activities and may possess strand displacement activity. Any such suitable polymerase enzyme may be used.
  • Such a DNA polymerase may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3 >5 ’ exo-), M-MuLV reverse transcriptase.
  • the DNA polymerase may also be a genetically engineered variant.
  • the DNA polymerase may be a genetically engineered variant of the native DNA polymerase from Thermococcus species 9°N, such as species 9°N-7.
  • One such example of a modified polymerase is Therminator IX DNA polymerase or Therminator X DNA polymerase available from New England BioLabs.
  • DNA polymerases include Deep Vent (exo-), Vent (Exo-), 9°N DNA polymerase, Therminator DNA polymerase, Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase, Sulfolobus DNA polymerase I, and Taq Polymerase.
  • any suitable enzyme may be used.
  • an RNA polymerase may be used. Any suitable RNA polymerase may be used.
  • the RNA polymerase may be T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase holoenzyme.
  • any of the methods described herein it may be desirable to perform one or more additional method steps to extend one or both strands of the acceptor polynucleotide as part of the process of extending the acceptor polynucleotide by the methods of the invention, e.g. before, during or after a process of extending the acceptor polynucleotide using the ligase-mediated methods of the invention. It may be desirable to extend one or both strands as part of a double stranded acceptor polynucleotide. It may also be desirable to extend one or both strands as single stranded polynucleotides following separation of the two strands of the acceptor polynucleotide.
  • the enzyme may have a terminal transferase activity, e.g. the enzyme may be a terminal nucleotidyl transferase, or terminal deoxynucleotidyl transferase, and wherein the acceptor polynucleotide is extended to form a polynucleotide molecule comprising DNA or RNA, preferably DNA. Any of these enzymes may be used in the methods of the invention wherein such extension of an acceptor polynucleotide is required.
  • TdT terminal deoxynucleotidyl transferase
  • TdT terminal deoxynucleotidyl transferase
  • Pol lambda and pol micro enzymes may also be used (Ramadan K, et al., J. Mol. Biol., 2004, 339(2), 395-404), as may 029 DNA polymerase.
  • Directed evolution techniques conventional screening, rational or semi-rational engineering/mutagenesis methods or any other suitable methods may be used to alter any such enzyme to provide and/or optimise the required function.
  • Any other enzyme which is capable of extending a single-stranded polynucleotide molecule portion, such as a molecule comprising DNA or RNA, or one strand of a blunt-ended molecule with a nucleotide without the use of a template may be used.
  • a single stranded portion of a polynucleotide comprising DNA or blunt-ended double-stranded polynucleotide comprising DNA may be extended by an enzyme which has template-independent enzyme activity, such as template-independent polymerase or transferase activity.
  • the enzyme may have nucleotidyl transferase enzyme activity, e.g. a deoxynucleotidyl transferase enzyme, such as terminal deoxynucleotidyl transferase (TdT), or an enzyme fragment, derivative, analogue or functional equivalent thereof.
  • a polynucleotide extended by the action of such an enzyme comprises DNA.
  • a single stranded portion of an acceptor polynucleotide comprising RNA, or blunt-ended double-stranded acceptor polynucleotide comprising RNA may be extended by an enzyme which has nucleotidyl transferase enzyme (e.g. including TdT), or an enzyme fragment, derivative, analogue or functional equivalent thereof.
  • An acceptor polynucleotide extended by the action of such an enzyme may comprise RNA.
  • any suitable nucleotidyl transferase enzyme may be used for the synthesis of a single stranded polynucleotide molecule comprising RNA, or a single stranded portion of a polynucleotide molecule comprising RNA.
  • Nucleotidyl transferase enzymes such as poly (U) polymerase and poly(A) polymerase (e.g. from E. coli) are capable of template-independent addition of nucleoside monophosphate units to polynucleotide synthesis molecules. Any of these enzymes may be applied to methods described herein, as well as any enzyme fragment, derivative, analogue or functional equivalent thereof provided that the nucleotidyl transferase function is preserved in the enzyme. Directed evolution techniques, conventional screening, rational or semi-rational engineering/mutagenesis methods or any other suitable methods may be used to alter any such enzyme to provide and/or optimise the required function.
  • ligation may be achieved using any suitable means.
  • the ligation step will be performed by a ligase enzyme.
  • the ligase may be a T3 DNA ligase or a T4 DNA ligase
  • the enzyme may be human DNA ligase III, T3 DNA ligase, T4 DNA ligase, a T4 DNA ligase which has improved thermal stability compared to wild-type T4 DNA ligase, or a T4 DNA ligase which has improved salt tolerance compared to wild-type T4 DNA ligase.
  • the ligase may a blunt TA ligase.
  • a blunt TA ligase is available from New England BioLabs (NEB). This is a ready-to-use master mix solution of T4 DNA Ligase, ligation enhancer, and optimized reaction buffer specifically formulated to improve ligation.
  • the enzyme is a T3 DNA ligase or a T4 DNA ligase which has improved salt tolerance compared to wild-type T4 DNA ligase.
  • Molecules, enzymes, chemicals and methods for ligating (joining) single- and double-stranded polynucleotides are well known to the skilled person.
  • the polynucleotide having a predefined sequence synthesised according to the methods described herein is double-stranded.
  • the synthesised polynucleotide overall is double-stranded and wherein the first strand is attached to the second strand by hybridization. Mismatches and regions of non-hybridization may be tolerated, provided that overall the first strand is attached to the second strand by hybridization.
  • the strands may be separated as required to form single-stranded molecules.
  • Hybridisation may be defined by moderately stringent or stringent hybridisation conditions.
  • a moderately stringent hybridisation condition uses a prewashing solution containing 5x sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridisation buffer of about 50% formamide, 6xSSC, and a hybridisation temperature of 55° C (or other similar hybridisation solutions, such as one containing about 50% formamide, with a hybridisation temperature of 42° C), and washing conditions of 60° C, in 0.5xSSC, 0.1% SDS.
  • a stringent hybridisation condition hybridises in 6xSSC at 45° C, followed by one or more washes in O.lxSSC, 0.2% SDS at 68° C.
  • the double-stranded polynucleotide having a predefined sequence synthesised according to the methods described herein may be retained as a double-stranded polynucleotide.
  • the two strands of the double-stranded polynucleotide may be separated to provide a single-stranded polynucleotide having a predefined sequence.
  • Conditions that permit separation of two strands of a double-stranded polynucleotide are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley- Interscience, New York (1995)).
  • the double-stranded polynucleotide having a predefined sequence synthesised according to the methods described herein may be amplified following synthesis. Any region of the double-stranded polynucleotide may be amplified. The whole or any region of the double-stranded polynucleotide may be amplified. Conditions that permit amplification of a double-stranded polynucleotide are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley- Interscience, New York (1995)).
  • any of the synthesis methods described herein may further comprise an amplification step wherein the double-stranded polynucleotide having a predefined sequence, or any region thereof, is amplified as described above.
  • Amplification may be performed by any suitable method, such as polymerase chain reaction (PCR), polymerase spiral reaction (PSR), loop mediated isothermal amplification (LAMP), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3 SR), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), helicase dependant amplification (HD A), ramification amplification method (RAM), recombinase polymerase amplification (RPA) etc.
  • PCR polymerase chain reaction
  • PSR polymerase spiral reaction
  • LAMP loop mediated isothermal amplification
  • NASBA nucleic acid sequence based amplification
  • NASBA self-sus
  • the first and/or second strands of the acceptor polynucleotide at the second terminal end may consist of a polynucleotide sequence which is complementary to the polynucleotide sequence of a first primer oligonucleotide.
  • the first primer oligonucleotide may be used to prime an amplification reaction to amplifiy all or a portion of the double-stranded polynucleotide having a predefined sequence.
  • the first primer oligonucleotide may be used together with a second primer oligonucleotide to prime the amplification reaction to amplifiy all or a portion of the double-stranded polynucleotide having a predefined sequence.
  • the second primer oligonucleotide consists of a polynucleotide sequence which is complementary to the polynucleotide sequence of a portion of the double-stranded polynucleotide having a predefined sequence to be amplified.
  • the first and second primer oligonucleotides bind to different sites on the double-stranded polynucleotide having a predefined sequence to be amplified, threby allowing amplicons of any desired length to be generated.
  • the amplification reaction may be any suitable amplification reaction, such as PCR. Amplicons generated from the amplification reaction may consequently be released from the template polynucleotide, which may remain tethered to a surface.
  • the double-stranded or single-stranded polynucleotide having a predefined sequence synthesised according to the methods described herein can be any length.
  • the polynucleotides can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450 or at least 500 nucleotides or nucleotide pairs in length.
  • the polynucleotides may be from about 10 to about 100 nucleotides or nucleotide pairs, about 10 to about 200 nucleotides or nucleotide pairs, about 10 to about 300 nucleotides or nucleotide pairs, about 10 to about 400 nucleotides or nucleotide pairs and about 10 to about 500 nucleotides or nucleotide pairs in length.
  • the polynucleotides can be up to about 1000 or more nucleotides or nucleotide pairs, up to about 5000 or more nucleotides or nucleotide pairs in length or up to about 100000 or more nucleotides or nucleotide pairs in length.
  • RNA molecules may be adapted for the synthesis of RNA.
  • the donor polynucleotide may be provided as an RNA/DNA hybrid polynucleotide. More specifically, the first strand of the donor polynucleotide may be provided as RNA and the second strand of the donor polynucleotide hybridised to the first strand may be provided as DNA. Following cycles of synthesis the resulting synthetic polynucleotide will itself be an RNA/DNA hybrid polynucleotide. Following synthesis it is then possible to separate the RNA and DNA strands of the hybrid polynucleotide. The DNA strand can be separated and the RNA strand can be retained for further use, e.g. in single-stranded form.
  • Synthetic polynucleotides produced in accordance with the synthesis methods of the invention may preferably be synthesised using solid phase or reversible solid phase techniques. A variety of such techniques is known in the art and may be used.
  • acceptor polynucleotides may be immobilized to a surface e.g. a planar surface such as glass, a gel-based material, or the surface of a microparticle such as a bead or functionalised quantum dot.
  • the material comprising the surface may itself be bound to a substrate.
  • acceptor polynucleotides may be immobilized to a gel-based material such as e.g. polyacrylamide, and wherein the a gel-based material is bound to a supporting substrate such as glass.
  • Polynucleotides may be immobilized or tethered to surfaces directly or indirectly. For example they may be attached directly to surfaces by chemical bonding. They may be indirectly tethered to surfaces via an intermediate surface, such as the surface of a microparticle or bead e.g. as in SPRI or as in electrowetting systems, as described below. Cycles of synthesis may then be initiated and completed whilst the acceptor polynucleotide incorporating the newly-synthesised polynucleotide is immobilized.
  • a double-stranded acceptor polynucleotide may be immobilized to a surface prior to the incorporation of the first payload.
  • Such an immobilized doublestranded acceptor polynucleotide may therefore act as an anchor to tether the doublestranded polynucleotide of the predefined sequence to the surface during and after synthesis.
  • both strands of a double-stranded acceptor polynucleotide may each be immobilized to the surface at the same end of the molecule.
  • a double-stranded acceptor polynucleotide may be provided with each strand connected at adjacent ends, such as via a hairpin loop at the second terminal end, i.e. the opposite end to the site of initiation of new synthesis, and connected ends may be immobilized on a surface.
  • synthetic acceptor polynucleotides Before initiating synthesis of a new double-stranded polynucleotide of predefined sequence synthetic acceptor polynucleotides can be synthesised by methods known in the art, including those described herein, and tethered to a surface.
  • Pre-formed polynucleotides can be tethered to surfaces by methods commonly employed to create nucleic acid microarrays attached to planar surfaces. For example, acceptor polynucleotides may be created and then spotted or printed onto a planar surface. Acceptor polynucleotides may be deposited onto surfaces using contact printing techniques. For example, solid or hollow tips or pins may be dipped into solutions comprising pre-formed acceptor polynucleotides and contacted with the planar surface. Alternatively, oligonucleotides may be adsorbed onto micro-stamps and then transferred to a planar surface by physical contact.
  • Non-contact printing techniques include thermic printing or piezoelectric printing wherein sub-nanolitre size microdroplets comprising pre-formed acceptor polynucleotides may be ejected from a printing tip using methods similar to those used in inkjet and bubblejet printing.
  • Single-stranded oligonucleotides may be synthesised directly on planar surfaces such as using so-called “on-chip” methods employed to create microarrays. Such single-stranded oligonucleotides may then act as attachment sites to immobilize preformed acceptor polynucleotides.
  • On-chip techniques for generating single-stranded oligonucleotides include photolithography which involves the use of UV light directed through a photolithographic mask to selectively activate a protected nucleotide allowing for the subsequent incorporation of a new protected nucleotide. Cycles of UV -mediated deprotection and coupling of pre-determined nucleotides allows the in situ generation of an oligonucleotide having a desired sequence.
  • oligonucleotides may be created on planar surfaces by the sequential deposition of nucleobases using inkjet printing technology and the use of cycles of coupling, oxidation and deprotection to generate an oligonucleotide having a desired sequence (for a review see Kosuri and Church, Nature Methods, 2014, 11, 499- 507).
  • surfaces can be made of any suitable material.
  • a surface may comprise silicon, glass or polymeric material.
  • a surface may comprise a gel surface, such as a polyacrylamide surface, such as about 2% polyacrylamide, optionally a polyacrylamide surface derived using N- (5- bromoacetamidylpentyl) acrylamide (BRAPA), preferably the polyacrylamide surface is coupled to a solid support, such as glass.
  • BRAPA N- (5- bromoacetamidylpentyl) acrylamide
  • Synthetic polynucleotides having a predefined sequence can be synthesised in accordance with the invention using binding surfaces and structures, such as particles (e.g. microparticles) and beads, which facilitate reversible immobilization.
  • Solid phase reversible immobilization (SPRI) methods or modified methods are known in the art and may be employed (e.g. see DeAngelis M. M. et al. (1995) Solid-Phase Reversible Immobilization for the Isolation of PCR Products, Nucleic Acids Research, 23(22): 4742-4743.).
  • Surfaces can be provided in the form of microparticles, such as paramagnetic beads.
  • Paramagnetic beads can agglomerate under the influence of a magnetic field.
  • paramagnetic surfaces can be provided with chemical groups, e.g. carboxyl groups, which in appropriate attachment conditions will act as binding moieties for nucleic acids, as described in more detail below. Nucleic acids can be eluted from such surfaces in appropriate elution conditions.
  • Surfaces of microparticles and beads can be provided with UV-sensitive polycarbonate. Nucleic acids can be bound to the activated surface in the presence of a suitable immobilization buffer.
  • Microparticles and beads may be allowed to move freely within a reaction solution and then reversibly immobilized, e.g. by holding the bead within a microwell or pit etched into a surface.
  • a bead can be localised as part of an array e.g. by the use of a unique nucleic acid “barcode” attached to the bead or by the use of colour-coding.
  • acceptor polynucleotides in accordance with the invention can be synthesised and then reversibly immobilized to such binding surfaces.
  • Polynucleotides synthesised by methods of the invention can be synthesised whilst reversibly immobilized to such binding surfaces.
  • the surface may be part of an electro wetting-on-dielectric system (EWOD).
  • EWOD systems provide a dielectric-coated surface which facilitates microfluidic manipulation of very small liquid volumes in the form of microdroplets (e.g. see Chou, W-L., et al. (2015) Recent Advances in Applications of Droplet Micro fluidics, Micromachines, 6: 1249-1271.).
  • Droplet volumes can programmably be created, moved, partitioned and combined on-chip by electrowetting techniques.
  • electrowetting systems provide alternative means to reversibly immobilize polynucleotides during and after synthesis.
  • Polynucleotides having a predefined sequence may be synthesised in solid phase by methods described herein, wherein polynucleotides are immobilized on an EWOD surface and required steps in each cycle facilitated by electrowetting techniques.
  • electrowetting techniques For example, reagents required for each step, as well as for any required washing steps to remove used and unwanted reagent, can be provided in the form of microdroplets transported under the influence of an electric field via electrowetting techniques.
  • microfluidic platforms are available which may be used in the synthesis methods of the invention.
  • the emulsion-based microdroplet techniques which are commonly employed for nucleic acid manipulation can be used.
  • microdroplets are formed in an emulsion created by the mixing of two immiscible fluids, typically water and an oil.
  • Emulsion microdroplets can be programmably be created, moved, partitioned and combined in micro fluidic networks.
  • Hydrogel systems are also available.
  • microdroplets may be manipulated in any suitable compatible system, such as EWOD systems described above and other microfluidic systems, e.g. microfluidic systems comprising architectures based on components comprising elastomeric materials.
  • Microdroplets may be of any suitable size, provided that they are compatible with the synthesis methods herein. Microdroplet sizes will vary depending upon the particular system employed and the relevant architecture of the system. Sizes may thus be adapted as appropriate. In any of the synthesis methods described herein droplet diameters may be in the range from about 150nm to about 5mm. Droplet diameters below 1 pm may be verified by means known in the art, such as by techniques involving capillary jet methods, e.g. as described in Ganan-Calvo et al. (Nature Physics, 2007, 3, pp737-742)
  • the intermediate products of synthesis or assembly, or the final polynucleotide synthesis products may be sequenced as a quality control check to determine whether the desired polynucleotide or polynucleotides have been correctly synthesised or assembled.
  • the polynucleotide or polynucleotides of interest can be removed from the solid phase synthesis platform and sequenced by any one of a number of known commercially available sequencing techniques such as nanopore sequencing using a MinlONTM device sold by Oxford Nanopore Technologies Ltd. In a particular example, the sequencing may be carried out on the solid phase platform itself, removing the need to transfer the polynucleotide to a separate synthesis device.
  • Sequencing may be conveniently carried out on the same electrowetting device, such as an EWOD device as used for synthesis whereby the synthesis device comprises one or more measurement electrode pairs.
  • a droplet comprising the polynucleotide of interest can be contacted with one of the electrodes of the electrode pair, the droplet forming a droplet interface bilayer with a second droplet in contact with the second electrode of the electrode pair wherein the droplet bilayer interface comprises a nanopore in an amphipathic membrane.
  • the polynucleotide can be caused to translocate the nanopore for example under enzyme control and ion current flow through the nanopore can be measured under a potential difference between the electrode pair during passage of the polynucleotide through the nanopore.
  • the ion current measurements over time can be recorded and used to determine the polynucleotide sequence.
  • the polynucleotide Prior to sequencing, the polynucleotide may be subjected to one or more sample preparation steps in order to optimise it for sequencing such as disclosed in patent application no. PCT/GB2015/050140.
  • sample preparation steps such as disclosed in patent application no. PCT/GB2015/050140.
  • Examples of enzymes, amphipathic membranes and nanopores which may be suitably employed are disclosed in patent application nos. PCT/GB2013/052767 and PCT/GB2014/052736.
  • the necessary reagents for sample preparation of the polynucleotide, nanopores, amphipathic membranes and so on may be supplied to the EWOD device via sample inlet ports.
  • the sample inlet ports may be connected to reagent chambers.
  • polynucleotides will typically be attached chemically, they may also be attached to surfaces by indirect means such as via affinity interactions.
  • polynucleotides may be functionalised with biotin and bound to surfaces coated with avidin or streptavidin.
  • avidin or streptavidin For the immobilization of polynucleotides to surfaces (e.g. planar surfaces), microparticles and beads etc., a variety of surface attachment methods and chemistries are available. Surfaces may be functionalised or derivatized to facilitate attachment. Such functionalisations are known in the art.
  • a surface may be functionalised with a polyhistidine-tag (hexa histidine -tag, 6xHis-tag, His6 tag or His- tag®), Ni-NTA, streptavidin, biotin, an oligonucleotide, a polynucleotide (such as DNA, RNA, PNA, GNA, TNA or LNA), carboxyl groups, quaternary amine groups, thiol groups, azide groups, alkyne groups, DIBO, lipid, FLAG-tag (FLAG octapeptide), polynucleotide binding proteins, peptides, proteins, antibodies or antibody fragments.
  • the surface may be functionalised with a molecule or group which specifically binds to the acceptor polynucleotide.
  • polynucleotides may be tethered to a common surface via one or more covalent bonds.
  • the one or more covalent bonds may be formed between a functional group on the common surface and a functional group on the polynucleotides molecule.
  • the functional group on the polynucleotide molecule may be e.g. an amine group, a thiol group, a thiophosphate group or a thioamide group.
  • the functional group on the common surface may be a bromoacetyl group, optionally wherein the bromoacetyl group is provided on a polyacrylamide surface derived using N- (5- bromoacetamidylpentyl) acrylamide (BRAPA).
  • a polynucleotide may be attached to a surface, either directly or indirectly, via a linker.
  • Any suitable linker which is biocompatible and hydrophilic in nature may be used.
  • a linker may be a linear linker or a branched linker.
  • a linker may comprise a hydrocarbon chain.
  • a hydrocarbon chain may comprise from 2 to about 2000 or more carbon atoms.
  • the hydrocarbon chain may comprise an alkylene group, e.g. C2 to about 2000 or more alkylene groups.
  • the hydrocarbon chain may have a general formula of -(CH2)n- wherein n is from 2 to about 2000 or more.
  • the hydrocarbon chain may be optionally interrupted by one or more ester groups (i.e. -C(O)-O-) or one or more amide groups (i.e. -C(O)-N(H)-).
  • Any linker may be used selected from the group comprising PEG, polyacrylamide, poly(2-hydroxyethyl methacrylate), Poly-2 -methyl-2-oxazoline (PMOXA), zwitterionic polymers, e.g. poly(carboxybetaine methacrylate) (PCBMA), poly[ N -(3 -sulfopropyl)- N -methacryloxyethyl- N , N dimethyl ammonium betaine] (PSBMA), glycopolymers, and polypeptides.
  • PCBMA poly(carboxybetaine methacrylate)
  • PSBMA poly[ N -(3 -sulfopropyl)- N -methacryloxyethyl- N , N dimethyl ammonium betaine]
  • a linker may comprise a polyethylene glycol (PEG) having a general formula of -(CH2-CH2-O)n-, wherein n is from 1 to about 600 or more.
  • PEG polyethylene glycol
  • a linker may comprise oligoethylene glycol-phosphate units having a general formula of -[(CH2-CH2-O)n-PO2'-O] m - where n is from 1 to about 600 or more and m could be 1-200 or more.
  • any of the above-described linkers may be attached at one end of the linker to an acceptor molecule as described herein, and at the other end of the linker to a first functional group wherein the first functional group may provide a covalent attachment to a surface.
  • the first functional group may be e.g. an amine group, a thiol group, a thiophosphate group or a thioamide group as further described herein.
  • the surface may be functionalised with a further functional group to provide a covalent bond with the first functional group.
  • the further functional group may be e.g. a 2 -bromoacetamido group as further described herein.
  • a bromoacetyl group is provided on a polyacrylamide surface derived using N- (5- bromoacetamidylpentyl) acrylamide (BRAPA).
  • the further functional group on the surface may be a bromoacetyl group, optionally wherein the bromoacetyl group is provided on a polyacrylamide surface derived using N- (5- bromoacetamidylpentyl) acrylamide (BRAPA) and the first functional group may be e.g. an amine group, a thiol group, a thiophosphate group or a thioamide group as appropriate.
  • the surface to which polynucleotides are attached may comprise a gel.
  • the surface comprises a polyacrylamide surface, such as about 2% polyacrylamide, preferably the polyacrylamide surface is coupled to a solid support such as glass.
  • an acceptor polynucleotide may optionally be attached to a linker via a branching nucleotide incorporated into the acceptor polynucleotide. Any suitable branching nucleotide may be used with any suitable compatible linker.
  • acceptor polynucleotides Prior to initiating synthesis cycles of the invention, acceptor polynucleotides may be synthesised with one or more branching nucleotides incorporated into the polynucleotide.
  • the exact position at which the one or more branching nucleotides are incorporated into the polynucleotide, and thus where a linker may be attached, may vary and may be chosen as desired. The position may e.g. be at the terminal end of a strand or e.g. in the loop region which connects first and second strands in embodiments which comprise a hairpin loop.
  • the one or more branching nucleotides may be incorporated into the acceptor polynucleotide with a blocking group which blocks a reactive group of the branching moiety.
  • the blocking group may then be removed (deblocked) prior to the coupling to the branching moiety of the linker, or a first unit (molecule) of the linker if a linker comprises multiple units.
  • the one or more branching nucleotides may be incorporated into the polynucleotide with a group suitable for use in a subsequent “click chemistry” reaction to couple to the branching moiety the linker, or a first unit of the linker if a linker comprises multiple units.
  • An example of such a group is an acetylene group.
  • a linker may optionally comprise one or more spacer molecules (units), such as e.g. an Sp9 spacer, wherein the first spacer unit is attached to the branching nucleotide.
  • the linker may comprise one or more further spacer groups attached to the first spacer group.
  • the linker may comprise multiple e.g. Sp9 spacer groups.
  • a first spacer group is attached to the branching moiety and then one or more further spacer groups are sequentially added to extend a spacer chain comprising multiple spacer units connected with phosphate groups therebetween. Shown below are some non-limiting examples of spacer units (Sp3, Sp9 and
  • Spl3 which could comprise the first spacer unit attached to a branching nucleotide, or a further spacer unit to be attached to an existing spacer unit already attached to the branching nucleotide.
  • a linker may comprise one or more ethylene glycol units.
  • a linker may comprise an oligonucleotide, wherein multiple units are nucleotides.
  • the term 5 is used to differentiate from the 5 ’ end of the nucleotide to which the branching moiety is attached, wherein 5 ’ has its ordinary meaning in the art.
  • 5 it is intended to mean a position on the nucleotide from which a linker can be extended.
  • the 5 ’ ’ position may vary.
  • the 5 ’ ’ position is typically a position in the nucleobase of the nucleotide.
  • the 5 ’ ’ position in the nucleobase may vary depending on the nature of the desired branching moiety, as depicted in the structures above.
  • the first and second strands of the acceptor polynucleotide at the second terminal end may each be tethered to a surface;
  • the first and second strands of the acceptor polynucleotide at the second terminal end may connected together by a polynucleotide hairpin loop and tethered to a surface;
  • the first strand of the acceptor polynucleotide at the second terminal end may be tethered to a surface and the second strand of the acceptor polynucleotide at the second terminal end may be untethered;
  • the second strand of the acceptor polynucleotide at the second terminal end may be tethered to a surface and the first strand of the acceptor polynucleotide at the second terminal end may be untethered.
  • the tethered strand(s) at the second terminal end may comprise a cleavable linker(s), wherein the linker(s) may be cleaved to detach the double-stranded polynucleotide from the surface following synthesis.
  • the hairpin loop may be tethered to a surface via a cleavable linker, wherein the linker may be cleaved to detach the double-stranded polynucleotide from the surface following synthesis.
  • the cleavable linker may be a UV cleavable linker.
  • the tethered strand(s) at the second terminal end may include a recognition site, which may be used to cleave the double-stranded polynucleotide and thereby detach the double-stranded polynucleotide from the surface following synthesis.
  • the recognition site may be a recognition site for an endonuclease.
  • the recognition site may be a recognition site for a restriction enzyme.
  • the recognition site may comprise a cleavage site defined by a uracil nucleotide positioned in either the first or second strands of the acceptor polynucleotide, wherein cleavage may be performed by an enzyme having uracil DNA glycosylase activity and DNA glycosylase-lyase activity e.g. Endonuclease VIII activity.
  • any of the polynucleotide synthesis methods described herein may be used to manufacture a polynucleotide microarray (Trevino, V. et al., Mol. Med. 2007 13, pp527-541).
  • acceptor polynucleotides may be tethered to a plurality of individually addressable reaction sites on a surface and polynucleotides having a predefined sequence may be synthesised in situ on the microarray.
  • the polynucleotide of predefined sequence may be provided with a unique sequence.
  • the polynucleotides may be provided with barcode sequences to facilitate identification.
  • microarray manufacture may be performed using techniques commonly used in this technical field, including techniques described herein.
  • acceptor polynucleotides may be tethered to surfaces using known surface attachment methods and chemistries, including those described herein.
  • Polynucleotides of predefined sequence may be provided at reaction sites in double-stranded form. Alternatively, following synthesis double-stranded polynucleotides may be separated and one strand removed, leaving single-stranded polynucleotides at reaction sites. Selective tethering of strands may be provided to facilitate this process as described elsewhere herein. Separation of strands may be performed by conventional methods, such as heat treatment.
  • a polynucleotide having a predefined sequence synthesised by methods described herein, and optionally amplified by methods described herein, may be joined to one or more other such polynucleotides to create larger synthetic polynucleotides.
  • Joining of multiple polynucleotides can be achieved by techniques commonly known in the art.
  • a first polynucleotide and one or more additional polynucleotides synthesised by methods described herein may be cleaved to create compatible termini and then polynucleotides joined together by ligation.
  • Cleavage can be achieved by any suitable means.
  • restriction enzyme cleavage sites may be created in polynucleotides and then restriction enzymes used to perform the cleavage step, thus releasing the synthesised polynucleotides from any other undesirable polynucleotide sequence.
  • Cleavage sites could be designed as part of the synthesised polynucleotides.
  • cleavage sites could be created within the newly-synthesised polynucleotide as part of the predefined nucleotide sequence.
  • Assembly of polynucleotides is preferably performed using solid phase methods. For example, following synthesis a first polynucleotide may be subject to a single cleavage at a suitable position distal to the site of surface immobilization. The first polynucleotide will thus remain immobilized to the surface, and the single cleavage will generate a terminus compatible for joining to another polynucleotide. An additional polynucleotide may be subject to cleavage at two suitable positions to generate at each terminus a compatible end for joining to other polynucleotides, and at the same time releasing the additional polynucleotide from surface immobilization.
  • the additional polynucleotide may be compatibly joined with the first polynucleotide thus creating a larger immobilized polynucleotide having a predefined sequence and having a terminus compatible for joining to yet another additional polynucleotide.
  • iterative cycles of joining of preselected cleaved synthetic polynucleotides may create much longer synthetic polynucleotide molecules.
  • the order of joining of the additional polynucleotides will be determined by the required predefined sequence.
  • assembly methods of the invention may allow the creation of synthetic polynucleotide molecules having lengths in the order of one or more Mb.
  • the assembly and/or synthesis methods of the invention may be performed using apparatuses known in the art. Techniques and apparatuses are available which allow very small volumes of reagents to be selectively moved, partitioned and combined with other volumes in different locations of an array, typically in the form of droplets Electrowetting techniques, such as electrowetting-on-dielectric (EWOD), may be employed, as described above. Suitable electrowetting techniques and systems that may be employed in the invention that are able to manipulate droplets are disclosed for example in US8653832, US8828336, US20140197028 and US20140202863.
  • EWOD electrowetting-on-dielectric
  • Cleavage from the solid phase may be achieved by providing cleavable linkers in one or both the primer strand portion and the portion of the support strand hybridized thereto.
  • the cleavable linker may be e.g. a UV cleavable linker.
  • polynucleotides having a predefined sequence may be synthesised whilst immobilized to an electrowetting surface, as described above.
  • Synthesised polynucleotides may be cleaved from the electrowetting surface and moved under the influence of an electric field in the form of a droplet.
  • Droplets may be combined at specific reaction sites on the surface where they may deliver cleaved synthesised polynucleotides for ligation with other cleaved synthesised polynucleotides.
  • Polynucleotides can then be joined, for example by ligation.
  • populations of different polynucleotides may be synthesised and attached in order according to the predefined sequence desired.
  • a fully automated polynucleotide synthesis and assembly system may be designed. The system may be programmed to receive a desired sequence, supply reagents, perform synthesis cycles and subsequently assemble desired polynucleotides according to the predefined sequence desired.
  • the invention also provides polynucleotide synthesis systems for carrying out any of the synthesis methods described and defined herein, as well as any of the subsequent amplification and assembly steps described and defined herein.
  • synthesis cycle reactions will be carried out by incorporating nucleotides of predefined sequence into acceptor polynucleotide molecules which are tethered to a surface by means described and defined herein.
  • the surface may be any suitable surface as described and defined herein.
  • reactions to incorporate nucleotides of predefined sequence into an acceptor polynucleotide molecule involve performing any of the synthesis methods on an acceptor polynucleotide within a reaction area.
  • a reaction area is any area of a suitable substrate to which an acceptor polynucleotide molecule is attached and wherein reagents for performing the synthesis methods may be delivered.
  • a reaction area may be a single area of a surface comprising a single acceptor polynucleotide molecule wherein the single acceptor polynucleotide molecule can be addressed with reagents.
  • a reaction area may be a single area of a surface comprising multiple acceptor polynucleotide molecules, wherein the acceptor polynucleotide molecules cannot be individually addressed with reagent in isolation from each other.
  • the multiple acceptor polynucleotide molecules in the reaction area are exposed to the same reagents and conditions and may thus give rise to synthetic polynucleotide molecules having the same or substantially the same nucleotide sequence.
  • a synthesis system for carrying out any of the synthesis methods described and defined herein may comprise multiple reaction areas, wherein each reaction area comprises one or more attached acceptor polynucleotide molecules and wherein each reaction area may be individually addressed with reagent in isolation from each of the other reaction areas.
  • Such a system may be configured e.g. in the form of an array, e.g. wherein reaction areas are formed upon a substrate, typically a planar substrate.
  • a system having a substrate comprising a single reaction area or comprising multiple reaction areas may be comprised within e.g. an EWOD system or a microfluidic system and the systems configured to deliver reagents to the reaction site.
  • EWOD and micro fluidic systems are described in more detail herein.
  • an EWOD system may be configured to deliver reagents to the reaction site(s) under electrical control.
  • a microfluidic system such as comprising microfabricated architecture e.g. as formed from elastomeric or similar material, may be configured to deliver reagents to the reaction site(s) under fluidic pressure and/or suction control or by mechanical means.
  • Reagents may be delivered by any suitable means, for example via carbon nanotubes acting as conduits for reagent delivery. Any suitable system may be envisaged.
  • EWOD, micro fluidic and other systems may be configured to deliver any other desired reagents to reaction sites, such as enzymes for cleaving a synthesised doublestranded polynucleotide from the acceptor polynucleotide following synthesis, and/or reagents for cleaving a linker to release an entire polynucleotide from the substrate and/or reagents for amplifying a polynucleotide molecule following synthesis or any region or portion thereof, and/or reagents for assembling larger polynucleotide molecules from smaller polynucleotide molecules which have been synthesised according to the synthesis methods of the invention.
  • enzymes for cleaving a synthesised doublestranded polynucleotide from the acceptor polynucleotide following synthesis and/or reagents for cleaving a linker to release an entire polynucleotide from the substrate and/or reagents for amp
  • kits for carrying out any of the synthesis methods described and defined herein.
  • a kit may contain any desired combination of reagents for performing any of the synthesis and/or assembly methods of the invention described and defined herein.
  • a kit may comprise any one or more volume(s) of reaction reagents comprising acceptor polynucleotides, donor polynucleotides, volume(s) of reaction reagents corresponding to any one or more steps of the synthesis cycles described and defined herein, volume(s) of reaction reagents comprising nucleotides comprising reversible blocking groups or reversible terminator groups, volume(s) of reaction reagents for amplifying one or more polynucleotide molecules following synthesis or any region or portion thereof, volume(s) of reaction reagents for assembling larger polynucleotide molecules from smaller polynucleotide molecules which have been synthesised according to the synthesis methods of the invention, volume(s) of reaction reagents for assembling larger
  • kits may contain any number of, and type of donor polynucleotides sufficient for the synthesis of a polynucleotide molecules of any given predetermined sequence, including mixtures of any desired length and sequence of polynucleotide payload, as required by the user.
  • Polynucleotide molecules are naturally capable of storing information encoded within them due to differences in the identity and sequences of nucleobases forming the structure of the polynucleotide molecule.
  • the natural data storage function of polynucleotide molecules can be exploited for the storage of new information by synthesising new polynucleotide molecules according to a specific nucleobase sequence which can thus encode new information within the polynucleotide molecule which can later be accessed or “read” to retrieve the information.
  • New information can, for example, be encoded into a polynucleotide molecule in a digital form.
  • the invention additionally provides methods of storing data in digital form in a polynucleotide molecule, thereby generating a nucleotide sequence in the polynucleotide synthesis molecule indicative of the “0” or “1” state of a bit of digital information.
  • a nucleotide sequence can be incorporated into a polynucleotide synthesis molecule to be indicative of the “0” or “1” state of a bit of digital information in any suitable way. For example bits of digital information can be created using two different species of nucleotide.
  • a polynucleotide can be extended so as to generate an adenine (A) - thymine (T) pair in a first cycle of synthesis followed by extension so as to generate a cytosine (C) - guanine (G) pair in a second subsequent cycle.
  • A-T pair in the polynucleotide molecule can thus be indicative of the “0” or “1” state of a bit of digital information.
  • the presence of the C-G pair juxtaposed adjacent to the A-T pair can thus be indicative of the opposite state of the bit.
  • A-T and C-G pairs of nucleobases in sequence can therefore allow for digital information to be encoded into the polynucleotide in bit form.
  • A-T and C-G are provided as examples only. Any nucleobases can be used provided they can be distinguished from each other.
  • Bits can alternatively be generated by the incorporation of two or more, i.e. a first string, of nucleobases of the same or indistinguishable species in the same or successive cycles of synthesis which can thus be indicative of the “0” or “1” state of a bit of digital information. This can then be followed by the incorporation of two or more, i.e. a second string, of nucleobases of the same or indistinguishable species in the same or successive cycles of synthesis which can thus be indicative of the opposite state of the bit to that previously generated.
  • nucleobases can be used provided that the nucleobases of the first string can be distinguished from the nucleobases of the second string.
  • First and second strings need not consist of the same number of nucleobases since the transition between first and second string is indicative of the transition between the “0” or “1” state of the bit of digital information and the opposite state of the bit.
  • Any such method of data storage may be performed using any of the in vitro methods of synthesising a double-stranded polynucleotide molecule as described and defined herein. Any such method of data storage may be performed using any of the apparatus, devices and systems described and defined herein.
  • the method may not involve a step of incorporation of a polynucleotide having a reversible terminator group (reversible blocking group) and an additional step of deprotection to remove the reversible terminator group.
  • a reversible terminator group reversible blocking group
  • a reversible terminator group is a chemical group which is incorporated into a nucleic acid strand and which acts to prevent further extension of the strand by an enzyme, such as a polymerase enzyme. Examples of reversible terminators are provided below.
  • Nitrobenzyl reversible terminators Disulfide reversible terminators: Azidomethyl reversible therminators:
  • Nucleoside triphosphates with bulky groups attached to the base can serve as substitutes for a reversible terminator group on 3 ’-hydroxy group and can block further incorporation guanine adenine
  • an acceptor polynucleotide is first provided.
  • the acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the acceptor polynucleotide is ligatable and blunt- ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end may or may not comprise a 3’ hydroxyl group.
  • the second terminal end of the acceptor polynucleotide is preferably non- ligatable.
  • the second terminal end of the acceptor polynucleotide is preferably tethered to a surface.
  • a surface may be any suitable surface as described and defined elsewhere herein.
  • the second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface.
  • the first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.
  • the acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesize. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include pairs of nucleotides of the predefined sequence at the first terminal end, for example where the acceptor polynucleotide is the product of a previous cycle of synthesis.
  • a donor polynucleotide is also provided.
  • the donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the donor polynucleotide is ligatable and blunt-ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end is typically also free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end may be non-ligatable or it may be ligatable (i.e. in the case of a symmetrical donor, as described below).
  • the terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide lacks a 5’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the second terminal end of the donor polynucleotide may be non-ligatable. This avoids the problem of multiple donor polynucleotides ligating together.
  • the terminal nucleotides of the first and/or second strands at the second terminal end may comprise a blocking group.
  • a blocking group is any blocking group defined elsewhere herein.
  • a blocking group(s) renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be provided without a 5’ phosphate group.
  • the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be ligatable with respect to an acceptor polynucleotide, but it may be structured so that it cannot ligate to another donor polynucleotide.
  • the donor polynucleotide may be a symmetrical donor polynucleotide.
  • the second terminal end of the donor polynucleotide may comprise a second polynucleotide payload, wherein:
  • the payload nucleotide sequence of the first strand at the second terminal end of the donor polynucleotide in the 5 ’ to 3 ’ direction is the same as the payload nucleotide sequence of the second strand at the first terminal end of the donor polynucleotide in the 5 ’ to 3 ’ direction;
  • the payload nucleotide sequence of the second strand at the second terminal end of the donor polynucleotide in the 3 ’ to 5 ’ direction is the same as the payload nucleotide sequence of the first strand at the first terminal end of the donor polynucleotide in the 3 ’ to 5 ’ direction;
  • the second terminal end of the donor polynucleotide comprises a 3 ’ hydroxyl group and lacks a 5’ phosphate group.
  • both the first and the second terminal ends lack 5 ’ phosphate groups. Accordingly, a first donor polynucleotide cannot be ligated to a second donor polynucleotide in a standard ligation reaction, thus avoiding the problem of self-ligation between donor polynucleotides. Nevertheless, since both the first and the second terminal ends comprise a 3’ hydroxyl group, either end of the donor polynucleotide is capable of ligating to an acceptor polynucleotide as described below. Such a donor polynucleotide therefore has the advantage of possessing two identical ligatable (with respect to an acceptor polynucleotide) ends. By having more ligatable ends available per reaction, the efficiency of ligation can be improved.
  • the donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence.
  • the polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation.
  • the terminal nucleotide of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload.
  • the penultimate nucleotide of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.
  • the donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload.
  • the cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide.
  • the exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below.
  • the ligation step functions to physically join the donor polynucleotide to the acceptor polynucleotide as a first step to facilitate the transfer of the polynucleotide payload from the donor polynucleotide to the acceptor polynucleotide.
  • the ligation step comprises ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide.
  • the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.
  • the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end, and wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick.
  • the ligation step comprises a single-stranded ligation wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the second strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.
  • the ligation step can be performed by any suitable means for physically joining together polynucleotide strands that were previously not joined together.
  • the ligation step is performed by the action of an enzyme having nucleotide ligase activity, such as any ligase enzyme described elsewhere herein and which can perform the required ligase function for this particular method version.
  • the step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.
  • Acceptor depletion (optional)
  • a depletion step may be performed before the cleavage step. Such a step is optional and not essential.
  • the depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide.
  • Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide.
  • Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.
  • an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.
  • a depletion step may comprise performing a treatment step to remove the 5 ’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide.
  • Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • an enzyme having phosphatase activity such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step.
  • the unreacted acceptor polynucleotide may act as a substrate for a nuclease enzyme, which can act to render an unreacted acceptor polynucleotide inert by degrading the first strand of the unreacted acceptor polynucleotide. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, an unreacted acceptor polynucleotide can act as a substrate for an enzyme having 5’ to 3’ exonuclease activity, which can degrade the first strand.
  • a depletion step may comprise performing a nuclease treatment step to degrade the first strand of the acceptor polynucleotide.
  • a depletion step may comprise performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity to degrade the first strand of the acceptor polynucleotide.
  • the donor polynucleotide for use in that cycle of synthesis should be provided such that the second terminal end of the donor polynucleotide cannot act as a substrate for an enzyme having 5 ’ to 3 ’ exonuclease activity.
  • the cleavage step functions to cleave the ligated polynucleotide such that the one or more nucleotide pairs of the predefined sequence comprising the polynucleotide payload that was previously part of the donor polynucleotide becomes incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide.
  • the cleavage step functions to separate the polynucleotide payload from the remainder of the donor polynucleotide.
  • the remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.
  • each polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that the user wishes to synthesize. Successive cycles therefore provide for the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by successively joining together multiple polynucleotide payloads.
  • the cleavage step may comprise cleaving the ligated polynucleotide at sites in both the first and second strands of the donor polynucleotide.
  • the cleavage step may comprise cleaving the ligated polynucleotide at a site in only the first strand of the donor polynucleotide.
  • the cleavage step may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at the same relative position in each strand.
  • Such a cleavage step is consequently performed as a symmetrical cleavage reaction, so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, wherein initially all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.
  • the nucleotides of the polynucleotide payload of the second strand at the first terminal end of the cleaved acceptor polynucleotide are separated from the donor polynucleotide and initially remain attached to the cleaved acceptor polynucleotide.
  • the nucleotides of the polynucleotide payload of the second strand remain attached only via interaction (e.g. hydrogen bonding) with the nucleotides of the polynucleotide payload of the first strand.
  • the nucleotides of the polynucleotide payload of the second strand are subsequently discarded.
  • the cleavage step may alternatively comprise cleaving the first strand of the ligated polynucleotide only.
  • a nick site is already present in the second strand.
  • Cleavage is performed in such a way that the first strand is cleaved at a different relative position compared to the nick site in the second strand.
  • Such a cleavage step consequently results in an asymmetrical cleavage.
  • the first strand is cleaved immediately above the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the donor polynucleotide).
  • the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide in the first strand, wherein the terminal nucleotide of the first strand of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • the second strand is cleaved below the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the acceptor polynucleotide).
  • method version 1 further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide. Accordingly, because the original nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage, they are consequently discarded. These steps generate the 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • nucleotides of the first strand of the polynucleotide payload overhang the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the polynucleotide payload.
  • method version 1 further comprises performing an incorporation step comprising extending the second strand of the acceptor polynucleotide by incorporating new payload nucleotides using the original payload nucleotides of the first strand in the overhang as templates, thereby re-forming the payload nucleotides in the second strand, thereby re-forming the payload nucleotide pairs in the ligated polynucleotide and thereby forming a ligatable blunt end at the first end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are incorporated at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides
  • cleavage of the first strand of the ligated polynucleotide is performed in such a way that a 5 ’ phosphate group is retained on the terminal nucleotide of the first strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the first strand. This is achieved as a consequence of any standard cleavage reaction.
  • incorporation is performed in such a way that a 3 ’ hydroxyl group is retained on the terminal nucleotide of the second strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the second strand.
  • method version 1 is performed such that following cleavage and following incorporation steps, the first terminal end of the cleaved acceptor polynucleotide comprising the polynucleotide payload is ligatable and is thus competent to be ligated to a further donor polynucleotide in the next cycle of synthesis.
  • the original nucleotides of the polynucleotide payload of the second strand remain attached only via interaction (e.g. hydrogen bonding) with the original nucleotides of the polynucleotide payload of the first strand.
  • the original nucleotides of the polynucleotide payload of the second strand may be separated from the first strand:
  • incorporation steps may be performed:
  • the cleavage step can be performed by any suitable means for creating the cleaved structures described above.
  • cleavage may comprise a double-stranded cleavage wherein both the first and second strands are cleaved.
  • Cleavage may comprise cleaving the sugar-phosphate backbone of the first and second strands of the donor polynucleotide molecule. In such a cleavage step both the first and second strands are cleaved at the same positions in a symmetric cleavage reaction. This generates a cleaved donor polynucleotide wherein the first terminal end is blunt-ended.
  • cleavage may be performed by a restriction enzyme.
  • cleavage may be performed by a type IIS restriction enzyme.
  • the type IIS restriction enzyme may be Mlyl.
  • the user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the required structure described above to be formed following cleavage.
  • cleavage may comprise a single-stranded cleavage wherein only the first strand is cleaved. Cleavage of only the first strand can be performed in view of the nick site introduced previously into the second strand.
  • the nucleotides of the second strand of the polynucleotide payload at the first terminal end of the donor polynucleotide remain attached to the donor polynucleotide at its first terminal end and are consequently discarded. These steps generate the 5 ’ overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • a single-stranded cleavage may comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.
  • Single-stranded cleavage may thus be performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, such as BspQI.
  • Single-stranded cleavage may alternatively be performed by the action of an enzyme having nicking cleavage function, preferably a type IIS restriction enzyme, optionally Nt.&/?01.
  • Single-stranded cleavage may alternatively be performed using a method wherein the cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide, wherein following cleavage the terminal nucleotide in the first strand of the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • the universal nucleotide may be inosine. Cleavage mechanisms using universal nucleotides are described elsewhere herein.
  • Single-stranded cleavage may alternatively be performed using a method wherein the cleavage site is defined by a uracil nucleotide and cleavage is performed by the combined action of a Uracil DNA glycosylase enzyme and a DNA glycosylase- lyase enzyme such as Endonuclease VIII.
  • the Uracil DNA glycosylase enzyme catalyses the excision of the uracil base, thus forming an abasic (apyrimidinic) site while at the same time leaving the phosphodiester backbone intact.
  • the DNA glycosylase-lyase enzyme activity creates a break in the phosphodiester backbone at the 3 ' and 5 ' sides of the abasic site, thus generating a single-strand break.
  • a new acceptor polynucleotide is created.
  • the new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with the polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide.
  • the new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.
  • an acceptor polynucleotide is first provided.
  • the acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the acceptor polynucleotide is ligatable and blunt- ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end may or may not comprise a 3’ hydroxyl group.
  • the second terminal end of the acceptor polynucleotide is preferably non- ligatable.
  • the second terminal end of the acceptor polynucleotide is preferably tethered to a surface.
  • a surface may be any suitable surface as described and defined elsewhere herein.
  • the second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface.
  • the first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.
  • the acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesize. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include pairs of nucleotides of the predefined sequence at the first terminal end, for example where the acceptor polynucleotide is the product of a previous cycle of synthesis.
  • a donor polynucleotide is also provided.
  • the donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the donor polynucleotide is ligatable and blunt-ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end is typically also free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end may be non-ligatable or it may be ligatable (i.e. in the case of a symmetrical donor, as described below).
  • the terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide lacks a 5’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the second terminal end of the donor polynucleotide may be non-ligatable. This avoids the problem of multiple donor polynucleotides ligating together.
  • the terminal nucleotides of the first and/or second strands at the second terminal end may comprise a blocking group.
  • a blocking group is any blocking group defined elsewhere herein.
  • a blocking group(s) renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be provided without a 5’ phosphate group.
  • the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be ligatable with respect to an acceptor polynucleotide, but it may be structured so that it cannot ligate to another donor polynucleotide.
  • the donor polynucleotide may be a symmetrical donor polynucleotide.
  • the second terminal end of the donor polynucleotide may comprise a second polynucleotide payload, wherein:
  • the payload nucleotide sequence of the first strand at the second terminal end of the donor polynucleotide in the 5 ’ to 3 ’ direction is the same as the payload nucleotide sequence of the second strand at the first terminal end of the donor polynucleotide in the 5 ’ to 3 ’ direction;
  • the payload nucleotide sequence of the second strand at the second terminal end of the donor polynucleotide in the 3 ’ to 5 ’ direction is the same as the payload nucleotide sequence of the first strand at the first terminal end of the donor polynucleotide in the 3 ’ to 5 ’ direction;
  • the second terminal end of the donor polynucleotide comprises a 3 ’ hydroxyl group and lacks a 5’ phosphate group.
  • both the first and the second terminal ends lack 5 ’ phosphate groups. Accordingly, a first donor polynucleotide cannot be ligated to a second donor polynucleotide in a standard ligation reaction, thus avoiding the problem of self-ligation between donor polynucleotides. Nevertheless, since both the first and the second terminal ends comprise a 3’ hydroxyl group, either end of the donor polynucleotide is capable of ligating to an acceptor polynucleotide as described below. Such a donor polynucleotide therefore has the advantage of possessing two identical ligatable (with respect to an acceptor polynucleotide) ends. By having more ligatable ends available per reaction, the efficiency of ligation can be improved.
  • the donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence.
  • the polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation.
  • the terminal nucleotide of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload.
  • the penultimate nucleotide of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.
  • the donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload.
  • the cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide.
  • the exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below.
  • the ligation step functions to physically join the donor polynucleotide to the acceptor polynucleotide as a first step to facilitate the transfer of the polynucleotide payload from the donor polynucleotide to the acceptor polynucleotide.
  • the ligation step comprises ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide.
  • the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.
  • the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end, and wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick.
  • the ligation step comprises a single-stranded ligation wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the second strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.
  • the ligation step can be performed by any suitable means for physically joining together polynucleotide strands that were previously not joined together.
  • the ligation step is performed by the action of an enzyme having nucleotide ligase activity, such as any ligase enzyme described elsewhere herein and which can perform the required ligase function for this particular method version.
  • the step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.
  • the method further comprises performing a first incorporation step to extend the second strand of the acceptor polynucleotide from the nick site.
  • the first incorporation step comprises synthesizing new nucleotides in the second strand using the nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesizing a new second strand of the donor polynucleotide and reforming the nucleotide pairs in the ligated polynucleotide including the one or more payload nucleotide pairs and the cleavage site.
  • the original second strand of the donor polynucleotide may be separated from the first strand:
  • incorporation steps may be performed: (a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the second strand when synthesising the new second strand; or
  • a depletion step may be performed before the cleavage step. Such a step is optional and not essential.
  • the depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide.
  • Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide.
  • Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.
  • an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.
  • a depletion step may comprise performing a treatment step to remove the 5 ’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide.
  • Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • an enzyme having phosphatase activity such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step.
  • the unreacted acceptor polynucleotide may act as a substrate for a nuclease enzyme, which can act to render an unreacted acceptor polynucleotide inert by degrading the first strand of the unreacted acceptor polynucleotide. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, an unreacted acceptor polynucleotide can act as a substrate for an enzyme having 5’ to 3’ exonuclease activity, which can degrade the first strand.
  • a depletion step may comprise performing a nuclease treatment step to degrade the first strand of the acceptor polynucleotide.
  • a depletion step may comprise performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity to degrade the first strand of the acceptor polynucleotide.
  • the donor polynucleotide for use in that cycle of synthesis should be provided such that the second terminal end of the donor polynucleotide cannot act as a substrate for an enzyme having 5 ’ to 3 ’ exonuclease activity.
  • the cleavage step functions to cleave the ligated polynucleotide such that the one or more nucleotide pairs of the predefined sequence comprising the polynucleotide payload, whether part of the original donor polynucleotide, or newly synthesized (as described further below), becomes incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide.
  • the cleavage step functions to separate the donor polynucleotide from the ligated polynucleotide.
  • the remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.
  • each polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that the user wishes to synthesize. Successive cycles therefore provide for the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by successively joining together multiple polynucleotide payloads.
  • the cleavage step comprises cleaving the ligated polynucleotide at sites in both the first and second strands of the donor polynucleotide.
  • the cleavage step may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at the same relative position in each strand.
  • Such a cleavage step is consequently performed as a symmetrical cleavage, so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, wherein all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.
  • cleavage is performed such that following cleavage the first terminal end of the acceptor polynucleotide comprising the polynucleotide payload is ligatable.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end i.e.
  • the second nucleotide of the final pair of nucleotides of the polynucleotide payload does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group. This is achieved as a consequence of any standard symmetrical cleavage reaction.
  • the cleavage step may alternatively comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at different relative positions in each strand.
  • Such a cleavage step is performed as an asymmetrical cleavage, so as to form a 5 ’overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide.
  • nucleotides of the first strand of the polynucleotide payload overhang the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the polynucleotide payload.
  • method version 2 further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide.
  • the first strand is cleaved immediately above the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the donor polynucleotide). Accordingly, the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide in the first strand, wherein the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • the second strand is cleaved below the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the acceptor polynucleotide). Accordingly, the nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage, and are consequently discarded. These steps generate the 5 ’ overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • method version 2 further comprises performing a second incorporation step comprising extending the second strand of the acceptor polynucleotide by incorporating new payload nucleotides using the payload nucleotides of the first strand in the overhang as templates, thereby re-forming the payload nucleotides in the second strand, thereby re-forming the payload nucleotide pairs in the ligated polynucleotide and thereby forming a ligatable blunt end at the first end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are incorporated at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleot
  • cleavage of the first strand of the ligated polynucleotide is performed in such a way that a 5 ’ phosphate group is retained on the terminal nucleotide of the first strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the first strand. This is achieved as a consequence of any standard cleavage reaction.
  • incorporation is performed in such a way that a 3’ hydroxyl group is retained on the terminal nucleotide of the second strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the second strand.
  • method version 2 is performed such that following cleavage and following incorporation steps, the first terminal end of the cleaved acceptor polynucleotide comprising the polynucleotide payload is ligatable and is thus competent to be ligated to a further donor polynucleotide in the next cycle of synthesis.
  • the cleavage step can be performed by any suitable means for creating the cleaved structures described above.
  • cleavage may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at the same relative position in each strand.
  • Such a cleavage step is consequently performed as a symmetrical cleavage, so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide.
  • Cleavage may comprise cleaving the sugar-phosphate backbone of the first and second strands of the donor polynucleotide molecule.
  • cleavage may be performed by a restriction enzyme.
  • cleavage may be performed by a type IIS restriction enzyme.
  • the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the required structure described above to be formed following cleavage.
  • cleavage may comprise an asymmetrical cleavage reaction, wherein the first strand of the ligated polynucleotide and the second strand of the ligated polynucleotide are cleaved at different relative positions in each strand.
  • the nucleotides of the second strand of the polynucleotide payload at the first terminal end of the donor polynucleotide remain attached to the donor polynucleotide at its first terminal end and are consequently discarded. These steps generate the 5 ’ overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • An asymmetrical cleavage reaction may comprises cleaving the sugar-phosphate backbone of the first and second strands of the ligated polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.
  • Asymmetrical cleavage may thus be performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, such as BspQI.
  • an enzyme having overhang cleavage function preferably a type IIS restriction enzyme, such as BspQI.
  • Asymmetrical cleavage may alternatively be performed using a method wherein the cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide, wherein following cleavage the terminal nucleotide in the first strand of the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • the universal nucleotide may be inosine. Cleavage mechanisms using universal nucleotides are described elsewhere herein.
  • Asymmetrical cleavage may alternatively be performed using a method wherein the cleavage site is defined by a uracil nucleotide and cleavage is performed by the combined action of a Uracil DNA glycosylase enzyme and a DNA glycosylase-lyase enzyme such as Endonuclease VIII.
  • the Uracil DNA glycosylase enzyme catalyses the excision of the uracil base, thus forming an abasic (apyrimidinic) site while at the same time leaving the phosphodiester backbone intact.
  • the DNA glycosylase-lyase enzyme activity creates a break in the phosphodiester backbone at the 3 ' and 5 ' sides of the abasic site, thus generating a single-strand break.
  • a new acceptor polynucleotide is created.
  • the new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with the polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide.
  • the new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.
  • an acceptor polynucleotide is first provided.
  • the acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the acceptor polynucleotide is ligatable and blunt- ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end may or may not comprise a 3’ hydroxyl group.
  • the second terminal end of the acceptor polynucleotide is preferably non- ligatable.
  • the second terminal end of the acceptor polynucleotide is preferably tethered to a surface.
  • a surface may be any suitable surface as described and defined elsewhere herein.
  • the second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface.
  • first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.
  • the acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesize. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include pairs of nucleotides of the predefined sequence at the first terminal end, for example where the acceptor polynucleotide is the product of a previous cycle of synthesis.
  • a donor polynucleotide is also provided.
  • the donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the donor polynucleotide is ligatable and blunt-ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end is typically also free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end is non-ligatable. This avoids the problem of multiple donor polynucleotides ligating together. Both polynucleotide strands of the second terminal end of the donor polynucleotide are connected together by a polynucleotide hairpin loop.
  • the terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide lacks a 5’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence.
  • the polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation.
  • the terminal nucleotide of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload.
  • the penultimate nucleotide of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.
  • the donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload.
  • the cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide.
  • the exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below.
  • the ligation step functions to physically join the donor polynucleotide to the acceptor polynucleotide as a first step to facilitate the transfer of the polynucleotide payload from the donor polynucleotide to the acceptor polynucleotide.
  • the ligation step comprises ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide.
  • the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.
  • the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end, and wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick.
  • the ligation step comprises a single-stranded ligation wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the second strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.
  • the ligation step can be performed by any suitable means for physically joining together polynucleotide strands that were previously not joined together.
  • the ligation step is performed by the action of an enzyme having nucleotide ligase activity, such as any ligase enzyme described elsewhere herein and which can perform the required ligase function for this particular method version.
  • the step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.
  • the method further comprises breaking the bonds between nucleotides of the first and second strands of the donor polynucleotide, thereby generating a single stranded donor polynucleotide ligated to the first strand of the acceptor polynucleotide, and synthesising a new second strand in the donor polynucleotide via a first incorporation step using the nucleotides of the first strand and original second strand as templates, preferably by the action of an enzyme having polymerase activity, thereby re-forming donor nucleotide pairs in the ligated polynucleotide including the one or more payload nucleotide pairs and the cleavage site.
  • the original second strand of the donor polynucleotide becomes part of the first strand, and a new second strand is created in the donor polynucleotide.
  • the polynucleotide hairpin loop which connects both polynucleotide strands of the second terminal end of the original donor polynucleotide may encode one strand of the cleavage site.
  • one strand of the cleavage site may be encoded in a sequence either side of the hairpin loop, or either side of and including a portion of the hairpin loop.
  • the step of synthesising a new second strand in the donor polynucleotide before the cleavage step comprises using the nucleotides of the first strand, the second strand and/or the hairpin in the donor polynucleotide as templates to generate a complete double-stranded cleavage site.
  • a depletion step may be performed before the cleavage step. Such a step is optional and not essential.
  • the depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide.
  • Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide.
  • Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.
  • an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.
  • a depletion step may comprise performing a treatment step to remove the 5 ’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide.
  • Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • an enzyme having phosphatase activity such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step.
  • the unreacted acceptor polynucleotide may act as a substrate for a nuclease enzyme, which can act to render an unreacted acceptor polynucleotide inert by degrading the first strand of the unreacted acceptor polynucleotide. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, an unreacted acceptor polynucleotide can act as a substrate for an enzyme having 5’ to 3’ exonuclease activity, which can degrade the first strand.
  • a depletion step may comprise performing a nuclease treatment step to degrade the first strand of the acceptor polynucleotide.
  • a depletion step may comprise performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity to degrade the first strand of the acceptor polynucleotide.
  • the donor polynucleotide for use in that cycle of synthesis should be provided such that the second terminal end of the donor polynucleotide cannot act as a substrate for an enzyme having 5 ’ to 3 ’ exonuclease activity.
  • the cleavage step functions to cleave the ligated polynucleotide such that the one or more nucleotide pairs of the predefined sequence comprising the polynucleotide payload, whether part of the original donor polynucleotide, or newly synthesized, becomes incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide.
  • the cleavage step functions to separate the donor polynucleotide from the ligated polynucleotide.
  • the remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.
  • each polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that the user wishes to synthesize. Successive cycles therefore provide for the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by successively joining together multiple polynucleotide payloads.
  • the cleavage step comprises cleaving the ligated polynucleotide at sites in both the first and second strands of the donor polynucleotide.
  • the cleavage step may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at the same relative position in each strand.
  • Such a cleavage step is consequently performed as a symmetrical cleavage, so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, wherein all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.
  • cleavage is performed such that following cleavage the first terminal end of the acceptor polynucleotide comprising the polynucleotide payload is ligatable.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end i.e.
  • the second nucleotide of the final pair of nucleotides of the polynucleotide payload does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group. This is achieved as a consequence of any standard symmetrical cleavage reaction.
  • the cleavage step may alternatively comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at different relative positions in each strand.
  • Such a cleavage step is performed as an asymmetrical cleavage, so as to form a 5 ’overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide.
  • nucleotides of the first strand of the polynucleotide payload overhang the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the polynucleotide payload.
  • method version 3 further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide.
  • the first strand is cleaved immediately above the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the donor polynucleotide). Accordingly, the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide in the first strand, wherein the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • the second strand is cleaved below the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the acceptor polynucleotide). Accordingly, the nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage, and are consequently discarded. These steps generate the 5 ’ overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • method version 3 further comprises performing a second incorporation step comprising extending the second strand of the acceptor polynucleotide by incorporating new payload nucleotides using the payload nucleotides of the first strand in the overhang as templates, thereby re-forming the payload nucleotides in the second strand, thereby re-forming the payload nucleotide pairs in the ligated polynucleotide and thereby forming a ligatable blunt end at the first end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are incorporated at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleot
  • cleavage of the first strand of the ligated polynucleotide is performed in such a way that a 5 ’ phosphate group is retained on the terminal nucleotide of the first strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the first strand. This is achieved as a consequence of any standard cleavage reaction.
  • incorporation is performed in such a way that a 3’ hydroxyl group is retained on the terminal nucleotide of the second strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the second strand.
  • method version 3 is performed such that following cleavage and following incorporation steps, the first terminal end of the cleaved acceptor polynucleotide comprising the polynucleotide payload is ligatable and is thus competent to be ligated to a further donor polynucleotide in the next cycle of synthesis.
  • cleavage step can be performed by any suitable means for creating the cleaved structures described above.
  • cleavage may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at the same relative position in each strand.
  • Such a cleavage step is consequently performed as a symmetrical cleavage, so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide.
  • Cleavage may comprise cleaving the sugar-phosphate backbone of the first and second strands of the donor polynucleotide molecule.
  • cleavage may be performed by a restriction enzyme.
  • cleavage may be performed by a type IIS restriction enzyme.
  • the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the required structure described above to be formed following cleavage.
  • cleavage may comprise an asymmetrical cleavage reaction, wherein the first strand of the ligated polynucleotide and the second strand of the ligated polynucleotide are cleaved at different relative positions in each strand.
  • the nucleotides of the second strand of the polynucleotide payload at the first terminal end of the donor polynucleotide remain attached to the donor polynucleotide at its first terminal end and are consequently discarded. These steps generate the 5 ’ overhang at the cleaved first terminal end of the acceptor polynucleotide.
  • An asymmetrical cleavage reaction may comprises cleaving the sugar-phosphate backbone of the first and second strands of the ligated polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.
  • Asymmetrical cleavage may thus be performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, such as BspQI.
  • an enzyme having overhang cleavage function preferably a type IIS restriction enzyme, such as BspQI.
  • Asymmetrical cleavage may alternatively be performed using a method wherein the cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide, wherein following cleavage the terminal nucleotide in the first strand of the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload.
  • the universal nucleotide may be inosine. Cleavage mechanisms using universal nucleotides are described elsewhere herein.
  • Asymmetrical cleavage may alternatively be performed using a method wherein the cleavage site is defined by a uracil nucleotide and cleavage is performed by the combined action of a Uracil DNA glycosylase enzyme and a DNA glycosylase-lyase enzyme such as Endonuclease VIII.
  • the Uracil DNA glycosylase enzyme catalyses the excision of the uracil base, thus forming an abasic (apyrimidinic) site while at the same time leaving the phosphodiester backbone intact.
  • the DNA glycosylase-lyase enzyme activity creates a break in the phosphodiester backbone at the 3 ' and 5 ' sides of the abasic site, thus generating a single-strand break.
  • a new acceptor polynucleotide is created.
  • the new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with the polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide.
  • the new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.
  • an acceptor polynucleotide is first provided.
  • the acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the acceptor polynucleotide is ligatable and blunt- ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end may or may not comprise a 3’ hydroxyl group.
  • the second terminal end of the acceptor polynucleotide is preferably non- ligatable.
  • the second terminal end of the acceptor polynucleotide is preferably tethered to a surface.
  • a surface may be any suitable surface as described and defined elsewhere herein.
  • the second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface.
  • the first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.
  • the acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesize. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include pairs of nucleotides of the predefined sequence at the first terminal end, for example where the acceptor polynucleotide is the product of a previous cycle of synthesis. Provision of donor polynucleotide
  • a donor polynucleotide is also provided.
  • the donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the donor polynucleotide is ligatable and blunt-ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end is typically also free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end may be non-ligatable or it may be ligatable (i.e. in the case of a symmetrical donor, as described below).
  • the terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide lacks a 5’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the second terminal end of the donor polynucleotide may be non-ligatable. This avoids the problem of multiple donor polynucleotides ligating together.
  • the terminal nucleotides of the first and/or second strands at the second terminal end may comprise a blocking group.
  • a blocking group is any blocking group defined elsewhere herein. A blocking group(s) renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be provided without a 5 ’ phosphate group.
  • the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be ligatable with respect to an acceptor polynucleotide, but it may be structured so that it cannot ligate to another donor polynucleotide.
  • the donor polynucleotide may be a symmetrical donor polynucleotide.
  • the second terminal end of the donor polynucleotide may comprise a second polynucleotide payload, wherein:
  • the payload nucleotide sequence of the first strand at the second terminal end of the donor polynucleotide in the 5 ’ to 3 ’ direction is the same as the payload nucleotide sequence of the second strand at the first terminal end of the donor polynucleotide in the 5 ’ to 3 ’ direction;
  • the payload nucleotide sequence of the second strand at the second terminal end of the donor polynucleotide in the 3 ’ to 5 ’ direction is the same as the payload nucleotide sequence of the first strand at the first terminal end of the donor polynucleotide in the 3 ’ to 5 ’ direction;
  • the second terminal end of the donor polynucleotide comprises a 3 ’ hydroxyl group and lacks a 5’ phosphate group.
  • both the first and the second terminal ends lack 5 ’ phosphate groups. Accordingly, a first donor polynucleotide cannot be ligated to a second donor polynucleotide in a standard ligation reaction, thus avoiding the problem of self-ligation between donor polynucleotides. Nevertheless, since both the first and the second terminal ends comprise a 3’ hydroxyl group, either end of the donor polynucleotide is capable of ligating to an acceptor polynucleotide as described below. Such a donor polynucleotide therefore has the advantage of possessing two identical ligatable (with respect to an acceptor polynucleotide) ends. By having more ligatable ends available per reaction, the efficiency of ligation can be improved.
  • the donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence.
  • the polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation.
  • the terminal nucleotide of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload.
  • the penultimate nucleotide of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.
  • the donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload.
  • the cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide.
  • the exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below.
  • the ligation step functions to physically join the donor polynucleotide to the acceptor polynucleotide as a first step to facilitate the transfer of the polynucleotide payload from the donor polynucleotide to the acceptor polynucleotide.
  • the ligation step comprises ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide.
  • the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.
  • the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end, and wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick.
  • the ligation step comprises a single-stranded ligation wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the second strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.
  • the step of joining the donor and acceptor polynucleotides at their first terminal ends e.g.
  • a second step of joining the donor and acceptor polynucleotides at their first terminal ends is performed.
  • a second ligation step may be performed comprising a second single-stranded ligation step wherein the second strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends.
  • the second strands of the donor and acceptor polynucleotides are ligated together by steps comprising adding a phosphate group to the second strand of the donor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide with the second strand of the acceptor polynucleotide.
  • PNK polynucleotide kinase
  • the ligation step can be performed by any suitable means for physically joining together polynucleotide strands that were previously not joined together.
  • the ligation step is performed by the action of an enzyme having nucleotide ligase activity, such as any ligase enzyme described elsewhere herein and which can perform the required ligase function for this particular method version.
  • the step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.
  • a depletion step may be performed before the cleavage step. Such a step is optional and not essential.
  • the depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide.
  • Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide.
  • Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.
  • an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.
  • a depletion step may comprise performing a treatment step to remove the 5 ’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide.
  • Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • an enzyme having phosphatase activity such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step.
  • the unreacted acceptor polynucleotide may act as a substrate for a nuclease enzyme, which can act to render an unreacted acceptor polynucleotide inert by degrading the first strand of the unreacted acceptor polynucleotide. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, an unreacted acceptor polynucleotide can act as a substrate for an enzyme having 5’ to 3’ exonuclease activity, which can degrade the first strand.
  • a depletion step may comprise performing a nuclease treatment step to degrade the first strand of the acceptor polynucleotide.
  • a depletion step may comprise performing a nuclease treatment step with an enzyme having 5 ’ to 3 ’ exonuclease activity to degrade the first strand of the acceptor polynucleotide.
  • the donor polynucleotide for use in that cycle of synthesis should be provided such that the second terminal end of the donor polynucleotide cannot act as a substrate for an enzyme having 5 ’ to 3 ’ exonuclease activity.
  • the cleavage step functions to cleave the ligated polynucleotide such that the one or more nucleotide pairs of the predefined sequence comprising the polynucleotide payload that was previously part of the donor polynucleotide becomes incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide.
  • the cleavage step functions to separate the polynucleotide payload from the remainder of the donor polynucleotide.
  • the remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.
  • each polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that the user wishes to synthesize. Successive cycles therefore provide for the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by successively joining together multiple polynucleotide payloads.
  • the cleavage step comprises cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide. Cleavage is performed so as to form, at the end of the cycle of synthesis, a blunt end at the cleaved first terminal end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.
  • cleavage is performed such that following cleavage the first terminal end of the acceptor polynucleotide comprising the polynucleotide payload is ligatable.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end i.e.
  • the second nucleotide of the final pair of nucleotides of the polynucleotide payload does not comprise a 5’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the cleavage step can be performed by any suitable means for creating the blunt-ended structure described above.
  • Cleavage may comprise a double-stranded cleavage reaction wherein both the first and second strands are cleaved. In such a cleavage step both the first and second strands are cleaved at the same positions in a symmetric cleavage reaction.
  • This generates a cleaved donor polynucleotide wherein the first terminal end is blunt-ended.
  • cleavage may be performed by a restriction enzyme.
  • cleavage may be performed by a type IIS restriction enzyme.
  • the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the blunt-ended structure described above to be formed following cleavage.
  • the cleavage step can also be performed via an asymmetrical cleavage reaction as described above for method versions 2 and 3, including any further incorporation steps required to reconstitute the polynucleotide payload and the blunt end at the first terminal end of the acceptor polynucleotide.
  • An asymmetrical cleavage reaction may be performed using any suitable reagents and methods as described for method versions 2 and 3 as described above.
  • a new acceptor polynucleotide is created.
  • the new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with the polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide.
  • the new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.
  • an acceptor polynucleotide is first provided.
  • the acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the acceptor polynucleotide is ligatable and blunt- ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide lacks a 5’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the second terminal end of the acceptor polynucleotide is preferably non- ligatable.
  • the second terminal end of the acceptor polynucleotide is preferably tethered to a surface.
  • a surface may be any suitable surface as described and defined elsewhere herein.
  • the second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface.
  • the first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.
  • the acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesize. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include pairs of nucleotides of the predefined sequence at the first terminal end, for example where the acceptor polynucleotide is the product of a previous cycle of synthesis.
  • a donor polynucleotide is also provided.
  • the donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the donor polynucleotide is ligatable and blunt-ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end is typically also free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end may be non-ligatable.
  • the terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide comprises a 5’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the second terminal end of the donor polynucleotide may be non-ligatable. This avoids the problem of multiple donor polynucleotides ligating together.
  • the terminal nucleotides of the first and/or second strands at the second terminal end may comprise a blocking group.
  • a blocking group is any blocking group defined elsewhere herein.
  • a blocking group(s) renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be provided without a 5 ’ phosphate group.
  • the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.
  • the donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence.
  • the polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation.
  • the terminal nucleotide of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload.
  • the penultimate nucleotide of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.
  • the donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload.
  • the cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide.
  • the exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below.
  • the ligation step functions to physically join the donor polynucleotide to the acceptor polynucleotide as a first step to facilitate the transfer of the polynucleotide payload from the donor polynucleotide to the acceptor polynucleotide.
  • the ligation step comprises ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide.
  • the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.
  • the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end, and wherein the first strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick.
  • the ligation step comprises a single-stranded ligation wherein the second strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the first strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.
  • a second step of joining the donor and acceptor polynucleotides at their first terminal ends is performed.
  • a second ligation step may be performed comprising a second single-stranded ligation step wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends.
  • the first strands of the donor and acceptor polynucleotides are ligated together by steps comprising adding a phosphate group to the first strand of the donor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide with the first strand of the acceptor polynucleotide.
  • PNK polynucleotide kinase
  • the ligation step can be performed by any suitable means for physically joining together polynucleotide strands that were previously not joined together.
  • the ligation step is performed by the action of an enzyme having nucleotide ligase activity, such as any ligase enzyme described elsewhere herein and which can perform the required ligase function for this particular method version.
  • the step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.
  • a depletion step may be performed before the cleavage step. Such a step is optional and not essential.
  • the depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide.
  • Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide.
  • Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.
  • an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.
  • a depletion step may comprise performing a treatment step to remove the 5 ’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide.
  • Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • an enzyme having phosphatase activity such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step.
  • the unreacted acceptor polynucleotide may act as a substrate for a nuclease enzyme, which can act to render an unreacted acceptor polynucleotide inert by degrading the first strand of the unreacted acceptor polynucleotide. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, an unreacted acceptor polynucleotide can act as a substrate for an enzyme having 5’ to 3’ exonuclease activity, which can degrade the first strand.
  • a depletion step may comprise performing a nuclease treatment step to degrade the first strand of the acceptor polynucleotide.
  • a depletion step may comprise performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity to degrade the first strand of the acceptor polynucleotide.
  • the donor polynucleotide for use in that cycle of synthesis should be provided such that the second terminal end of the donor polynucleotide cannot act as a substrate for an enzyme having 5 ’ to 3 ’ exonuclease activity.
  • the cleavage step functions to cleave the ligated polynucleotide such that the one or more nucleotide pairs of the predefined sequence comprising the polynucleotide payload that was previously part of the donor polynucleotide becomes incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide.
  • the cleavage step functions to separate the polynucleotide payload from the remainder of the donor polynucleotide.
  • the remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.
  • each polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that the user wishes to synthesize. Successive cycles therefore provide for the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by successively joining together multiple polynucleotide payloads.
  • the cleavage step comprises cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide. Cleavage is performed so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.
  • the cleavage step can be performed by any suitable means for creating the blunt-ended structure described above.
  • Cleavage may comprise a double-stranded cleavage reaction wherein both the first and second strands are cleaved. In such a cleavage step both the first and second strands are cleaved at the same positions in a symmetric cleavage reaction.
  • This generates a cleaved donor polynucleotide wherein the first terminal end is blunt-ended.
  • cleavage may be performed by a restriction enzyme.
  • cleavage may be performed by a type IIS restriction enzyme.
  • the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the blunt-ended structure described above to be formed following cleavage.
  • the cleavage step can also be performed via an asymmetrical cleavage reaction as described above for method versions 2 and 3, including any further incorporation steps required to reconstitute the polynucleotide payload and the blunt end at the first terminal end of the acceptor polynucleotide.
  • An asymmetrical cleavage reaction may be performed using any suitable reagents and methods as described for method versions 2 and 2 as described above.
  • the terminal nucleotide of the first strand at the first terminal end of the acceptor polynucleotide i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload
  • the terminal nucleotide of the first strand at the first terminal end of the acceptor polynucleotide comprises a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the first terminal end i.e. the second nucleotide of the final pair of nucleotides of the polynucleotide payload
  • the (3’) terminal nucleotide of the second strand at the first terminal end comprises a 3’ hydroxyl group.
  • the 5’ phosphate group joined to the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is removed, preferably by the action of an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • an enzyme having phosphatase activity such as calf intestinal phosphatase (CIP).
  • a new acceptor polynucleotide is created.
  • the new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with the polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide.
  • the new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.
  • an acceptor polynucleotide is first provided.
  • the acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the acceptor polynucleotide is ligatable and blunt- ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide lacks a 5’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the second terminal end of the acceptor polynucleotide is preferably non- ligatable.
  • the second terminal end of the acceptor polynucleotide is preferably tethered to a surface.
  • a surface may be any suitable surface as described and defined elsewhere herein.
  • the second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered.
  • the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface.
  • the first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.
  • the acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesize. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include pairs of nucleotides of the predefined sequence at the first terminal end, for example where the acceptor polynucleotide is the product of a previous cycle of synthesis.
  • a donor polynucleotide is also provided.
  • the donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends.
  • the first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.
  • the first terminal end of the donor polynucleotide is ligatable and blunt-ended.
  • the first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end is typically also free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.
  • the second terminal end may be non-ligatable.
  • the terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide comprises a 5’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end does not comprise a 5 ’ phosphate group.
  • the (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.
  • the second terminal end of the donor polynucleotide may be non-ligatable. This avoids the problem of multiple donor polynucleotides ligating together.
  • the terminal nucleotides of the first and/or second strands at the second terminal end may comprise a blocking group.
  • a blocking group is any blocking group defined elsewhere herein.
  • a blocking group(s) renders the second terminal end non-ligatable.
  • the second terminal end of the donor polynucleotide may be provided without a 5 ’ phosphate group.
  • the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.
  • the donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence.
  • the polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation.
  • the terminal nucleotide of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload.
  • the penultimate nucleotide of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.
  • the donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload.
  • the cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide.
  • the exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below. Ligation of acceptor and donor polynucleotides
  • the ligation step functions to physically join the donor polynucleotide to the acceptor polynucleotide as a first step to facilitate the transfer of the polynucleotide payload from the donor polynucleotide to the acceptor polynucleotide.
  • the ligation step comprises ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide.
  • the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.
  • the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end, and wherein the first strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick.
  • the ligation step comprises a single-stranded ligation wherein the second strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the first strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.
  • the ligation step can be performed by any suitable means for physically joining together polynucleotide strands that were previously not joined together.
  • the ligation step is performed by the action of an enzyme having nucleotide ligase activity, such as any ligase enzyme described elsewhere herein and which can perform the required ligase function for this particular method version.
  • the step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.
  • a further step is performed before the cleavage step.
  • the further step comprises performing an incorporation step to extend the first strand of the donor polynucleotide at its first terminal end at the nick site.
  • the step comprises synthesising new nucleotides in the first strand of the acceptor polynucleotide using the nucleotides of the second strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesising a new first strand of the acceptor polynucleotide and re-forming the nucleotide pairs between the first and second strands of the acceptor polynucleotide.
  • Such an incorporation step may be performed:
  • a depletion step may be performed before the cleavage step. Such a step is optional and not essential.
  • the depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide.
  • Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide.
  • Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.
  • an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, the unreacted acceptor polynucleotide can be rendered inert by removing the 5 ’ phosphate group so that it cannot be ligated to a donor polynucleotide in any further synthesis cycle.
  • a depletion step may comprise performing a treatment step to remove the 5 ’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide.
  • Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • CIP calf intestinal phosphatase
  • the unreacted acceptor polynucleotide may act as a substrate for a nuclease enzyme, which can act to render an unreacted acceptor polynucleotide inert by degrading the first strand of the unreacted acceptor polynucleotide. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, an unreacted acceptor polynucleotide can act as a substrate for an enzyme having 5’ to 3’ exonuclease activity, which can degrade the first strand.
  • a depletion step may comprise performing a nuclease treatment step to degrade the first strand of the acceptor polynucleotide.
  • a depletion step may comprise performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity to degrade the first strand of the acceptor polynucleotide.
  • the donor polynucleotide for use in that cycle of synthesis should be provided such that the second terminal end of the donor polynucleotide cannot act as a substrate for an enzyme having 5 ’ to 3 ’ exonuclease activity.
  • the cleavage step functions to cleave the ligated polynucleotide such that the one or more nucleotide pairs of the predefined sequence comprising the polynucleotide payload that was previously part of the donor polynucleotide becomes incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide.
  • the cleavage step functions to separate the polynucleotide payload from the remainder of the donor polynucleotide.
  • the remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.
  • each polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that the user wishes to synthesize. Successive cycles therefore provide for the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by successively joining together multiple polynucleotide payloads.
  • the cleavage step comprises cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide. Cleavage is performed so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.
  • the cleavage step can be performed by any suitable means for creating the blunt-ended structure described above.
  • Cleavage may comprise a double-stranded cleavage reaction wherein both the first and second strands are cleaved. In such a cleavage step both the first and second strands are cleaved at the same positions in a symmetric cleavage reaction.
  • This generates a cleaved donor polynucleotide wherein the first terminal end is blunt-ended.
  • cleavage may be performed by a restriction enzyme.
  • cleavage may be performed by a type IIS restriction enzyme.
  • the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the blunt-ended structure described above to be formed following cleavage.
  • the cleavage step can also be performed via an asymmetrical cleavage reaction as described above for method versions 2 and 3, including any further incorporation steps required to reconstitute the polynucleotide payload and the blunt end at the first terminal end of the acceptor polynucleotide.
  • An asymmetrical cleavage reaction may be performed using any suitable reagents and methods as described for method versions 2 and 3 as described above.
  • the terminal nucleotide of the first strand at the first terminal end of the acceptor polynucleotide i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload
  • the (3’) terminal nucleotide of the second strand at the first terminal end i.e. the second nucleotide of the final pair of nucleotides of the polynucleotide payload
  • the (3’) terminal nucleotide of the second strand at the first terminal end comprises a 3’ hydroxyl group.
  • the 5’ phosphate group joined to the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is removed, preferably by the action of an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).
  • an enzyme having phosphatase activity such as calf intestinal phosphatase (CIP).
  • a new acceptor polynucleotide is created.
  • the new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with the polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide.
  • the new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.
  • the strand of the acceptor polynucleotide shown in Figure 13 marked “5”’ corresponds to the first strand of the acceptor polynucleotide as described elsewhere herein.
  • the strand of the acceptor polynucleotide shown in Figure 13 marked “3”’ corresponds to the second strand of the acceptor polynucleotide as described elsewhere herein.
  • the terminal end of the acceptor polynucleotide shown in Figure 13 which is tethered to a surface corresponds with the second terminal end of the acceptor polynucleotide as described elsewhere herein.
  • the strand of the donor polynucleotide shown in Figure 13 marked “ligation strand” corresponds to the first strand of the donor polynucleotide as described elsewhere herein.
  • the strand of the donor polynucleotide shown in Figure 13 marked “helper strand” corresponds to the second strand of the donor polynucleotide as described elsewhere herein.
  • the terminal end of the donor polynucleotide shown in Figure 13 which comprises the payload corresponds with the first terminal end of the donor polynucleotide as described elsewhere herein.
  • the structure marked “payload” corresponds with the polynucleotide payload as described elsewhere herein.
  • Example 1 Surface immobilisation of acceptor DNA oligomers onto a 96 well plate and subsequent ligation of donor dsDNA
  • This example describes:
  • IX NEB Stick-Together ligation buffer 66 mM Tris-HCl, lOmM MgCh, 1 mM ATP, 7.5% PEG6000, 1 mM DTT, pH 7.6
  • ⁇ 1 uM salt- T4 DNA ligase NEB
  • 2 uM of donor dsDNA Table 2 (Ligation buffer mix).
  • Tris-EDTA buffer with IM NaCl and 0.05% (v/v) Tween 20 solution (TE-NaCl- Tween).
  • Example 2 Cleavage of acceptor-donor dsDNA complex and polymerase fill-in to deliver a NNN payload
  • This example describes:
  • Tris-EDTA buffer with IM NaCl and 0.05% (v/v) Tween 20 solution (TE-NaCl- Tween).
  • Example 3 Chemistry method version 1 with one-side blunt ligation, blunt cleavage, and incorporation.
  • This example describes the synthesis of polynucleotides using 3 steps: one-sided ligation of the payload onto double-stranded acceptor DNA, cleavage, and dNTP incorporation opposite the payload.
  • the first step takes place on a blunt-ended substrate with a 5’ phosphate group.
  • the method uses a donor, comprising of the ligation strand and the helper strand that facilitate ligation.
  • a schematic representation of the method is shown in Figure 4.
  • Binding and washing buffer 5 mM Tris-HCl, 0.5 mM EDTA, 1 M NaCl.
  • the resuspended beads (1 Ong/ pl) feature 40 pmol of immobilised acceptor.
  • the first step describes the ligation of polynucleotides with DNA ligase in the presence of a helper strand.
  • Oligonucleotides were designed in-house and synthesised by Integrated DNA Technologies (see Table 7 for sequences).
  • oligonucleotides Dilute the oligonucleotides to a stock concentration of 100 pM using sterile distilled water (ELGA VEOLIA).
  • reaction mixture by resuspending on a vortexer, centrifuged at 1,000 rpm for 5 seconds and incubate at room temperature for 30 minutes with gentle agitation.
  • Step 14 After the third wash, resuspend the bead pellet in 21.5 pl sterile water (ELGA VEOLIA) and continue with step 22 in section Step 2: Cleavage. Alternatively, resuspend the bead pellet in 8.6 pl sterile water and visualise the sample on a gel (steps 15 - 21).
  • Figure 14A shows the starting material which is Stul digested hairpin acceptor. It contains a mixture of molecules with a single 3’T-overhang (-75%) and blunt-ended molecules (-25%) that lost the overhang.
  • Figure 14B shows Stul-digested acceptor after ligation to double-stranded donor (lane 1). The ligation products are depleted following Mlyl cleavage (lane 2). Nucleotide incorporation by polymerase Q5 generates blunt-ended acceptor molecules, which yield ligation products in the presence of double-stranded donor (lane 3).
  • the second step describes a one-sided cleavage of the phosphodiester backbone above the payload.
  • a diagrammatic illustration is shown in Figure 4. Materials and Methods
  • Oligonucleotides were designed in-house and synthesised by Integrated DNA Technologies (see Table 7 for sequences).
  • oligonucleotides Dilute the oligonucleotides to a stock concentration of 100 pM using sterile distilled water (ELGA VEOLIA).
  • the third step describes the addition of dNTPs opposite the payload using enzymatic incorporation by a DNA polymerase.
  • a diagrammatic illustration is shown in Figure 4.
  • Oligonucleotides were designed in-house and synthesised by Integrated DNA Technologies (see Table 7 for sequences).
  • oligonucleotides were diluted to a stock concentration of 100 pM using sterile distilled water (ELGA VEOLIA).
  • Results showed that most starting molecules (molecules with either 3’T-overhangs or single-stranded payload overhangs) formed ligation products and thus were blunt-ended following Q5 polymerase extension (Figure 14B, lane 3).
  • This example describes the synthesis of polynucleotides using 5 steps: one-side blunt ligation between phosphorylated donor and second acceptor strand, phosphorylation of the 1 st acceptor strand, one-side ligation between donor and first acceptor strand, blunt cleavage above the payload and dephosphorylation.
  • a schematic representation of the method is shown in Figure 8.

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Abstract

L'invention concerne de nouveaux procédés de synthèse de molécules polynucléotidiques selon une séquence nucléotidique prédéfinie. L'invention concerne également des procédés d'assemblage de polynucléotides synthétiques après synthèse.
PCT/GB2024/051643 2023-06-27 2024-06-27 Procédé, kit et système de synthèse de polynucléotides Ceased WO2025003669A1 (fr)

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