WO2017013400A1 - Détecteur d'adn simple brin - Google Patents

Détecteur d'adn simple brin Download PDF

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WO2017013400A1
WO2017013400A1 PCT/GB2016/052124 GB2016052124W WO2017013400A1 WO 2017013400 A1 WO2017013400 A1 WO 2017013400A1 GB 2016052124 W GB2016052124 W GB 2016052124W WO 2017013400 A1 WO2017013400 A1 WO 2017013400A1
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protein
seq
suitably
amino acid
binding
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Martin Ronald Webb
Liisa Tellervo CHISTY
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Francis Crick Institute Ltd
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Francis Crick Institute Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
    • G01N2333/445—Plasmodium
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to sensor proteins for detection of single stranded DNA, for example in biological systems or solutions.
  • the sensor proteins are based on Plasm odium spp. single-stranded DNA binding protein (SSB).
  • Single-stranded DNA is a product of many cellular processes that include processing of dsDNA such as DNA replication, transcription, translation and repair. Single-stranded DNA may bind to multiple targets and form secondary structures, which may lead to undesirable events such as DNA mutation if left free in the cell. In nature, a ubiquitous protein has evolved to protect the single-stranded DNA, namely single-stranded DNA binding protein (SSB) and its structure and function is highly conserved between different bacterial species.
  • SSB single-stranded DNA binding protein
  • Fluorescent reagentless biosensors are single species that produce a fluorescent signal and can be used in situ to measure biomolecules 1 .
  • One way to develop reagentless biosensors is by using a protein scaffold to bind the molecule of interest specifically but also to be an adduct with the reporter fluorophore. Strengths of such reagentless biosensors are their simplicity, sensitivity and rapid response.
  • An existing reagentless biosensor for single-stranded DNA is an adduct of the tetrameric single-stranded DNA-binding protein (SSB) of Escherichia coli and a diethylaminocoumarin fluorophore 2 .
  • SSB tetrameric single-stranded DNA-binding protein
  • dsDNA double-stranded DNA
  • WO2008/152379 describes labelled single-strand DNA-binding protein (SSB) and its use in a biosensor for detection and visualisation of single-stranded DNA.
  • This document describes bacterial SSBs, in particular nine different bacterial SSBs in an alignment presented in Figure 8 of WO2008/152379.
  • Prior art sensor proteins such as labelled E.coli SSB proteins suffer from limited signal and/or sensitivity. It is a problem to provide sensor proteins with enhanced or increased signal and/ or sensitivity.
  • Prior art sensor proteins such as labelled E.coli SSB proteins have the drawback of decreasing fluorescence in real time assays. It is a problem to provide sensor proteins which do not suffer from this drawback.
  • a Plasm odium ssDNA binding protein is known. The Plasm odium ssDNA binding protein is very different to the E.coli ssDNA binding protein, including in terms of the amino acid sequence.
  • Prior art studies of the Plasm odium ssDNA binding protein have not attempted any labelling. Prior art studies have been carried out using intrinsic tryptophan residues only. These have been purely abstract studies not directed at the creation of any biosensor molecule.
  • the present invention seeks to overcome problem(s) associated with the prior art. SUMMARY OF THE INVENTION
  • Plasm odium SSB especially the exemplary Plasm odium falciparum SSB, a tetramer
  • the sensors of the invention have specific valuable properties which are absent from the art. For example, there is 20- fold fluorescence on DNA binding with PfSSB labelled at cysteine 93. Binding is rapid and may be diffusion controlled and 65-70 bases of DNA bind to each tetramer.
  • the sensor protein of the invention has only small changes in binding on changing salt conditions, which appears to be evidence that the 65-70-base binding mode predominates under a wide range of conditions. Thus the invention delivers a range of significant technical benefits over the known E.coli sensors.
  • the invention relates to a protein of the single stranded DNA binding domain (SSB) family, wherein said protein is a modified Plasm odium protein, said protein comprising at least one detectable label attached to an amino acid of said protein, wherein said amino acid is located on the (L1-1') loop of residues joining the two beta sheets ( ⁇ ) and ( ⁇ '), wherein the characteristics of the detectable label alter on binding single stranded DNA.
  • SSB single stranded DNA binding domain
  • the detectable label is attached to a region of the protein surface.
  • the detectable label is attached via a cysteine residue in the protein.
  • the cysteine residue is a naturally occurring cysteine residue.
  • the cysteine residue is, or corresponds to, C93 (Cys 93) of SEQ ID NO: 1.
  • cysteine residue is a cysteine residue engineered into the protein at a position which is, or corresponds to, one selected from the group consisting of: G92, E94 and K151 of SEQ ID NO: 1.
  • said modified Plasm odium protein is a modified Plasm odium falciparum protein.
  • said protein comprises amino acid sequence corresponding to at least amino acids 78 to 198 of SEQ ID NO:i,
  • amino acid sequence having at least 50% sequence identity to amino acids 78 to 198 of SEQ ID NO:i.
  • said protein comprises amino acid sequence corresponding to at least amino acids 78 to 250 of SEQ ID NO:i,
  • amino acid sequence having at least 50% sequence identity to amino acids 78 to 250 of SEQ ID NO:i.
  • said protein comprises amino acid sequence corresponding to at least amino acids 78 to 277 of SEQ ID NO:i,
  • amino acid sequence having at least 50% sequence identity to amino acids 78 to 277 of SEQ ID NO:i.
  • said protein comprises amino acid sequence corresponding to at least amino acids 78 to 285 of SEQ ID NO:i,
  • amino acid sequence having at least 50% sequence identity to amino acids 78 to 285 of SEQ ID NO:i.
  • polypeptide part of said protein consists of the amino acid sequence of SEQ ID NO:i.
  • the detectable label is a fluorescent label.
  • the detectable label is a coumarin.
  • the invention relates to a protein as described above wherein the label is selected from the group consisting of N-[2-(i-maleimidyl)ethyl]-7- diethylaminocoumarin-3-carboxamide and N-[2-(iodoacetamido)ethyl]-7- diethylaminocoumarin-3-carboxamide (IDCC).
  • the invention relates to a protein as described above wherein the label is IDCC.
  • the invention relates to a protein as described above which further comprises a mutation compared to SEQ ID NO: 1 at a position selected from the group consisting of K84, Y156, K187 and D189, preferably Y156.
  • a mutation is selected from the group consisting of K84D, Y156R, K187D and D189K, preferably Y156R.
  • the invention relates to a method for detecting single stranded DNA in a sample comprising the steps of:
  • the invention relates to a method for monitoring changes in ssDNA concentration in a sample comprising contacting said sample with a protein as described above and determining changes in the characteristics of the detectable label, wherein changes in the characteristics of the detectable label indicate changes in the concentration of ssDNA in said sample.
  • the characteristics of the detectable label are monitored by measurement of changes in fluorescence of a fluorophore comprised by said protein.
  • the invention relates to a method of screening for inhibitors of DNA processing enzymes which
  • the invention relates to use of a protein as described above in the determination of ssDNA concentration in a sample.
  • the invention relates to a nucleic acid having a nucleotide sequence encoding the polypeptide portion of the protein as described above.
  • proteins of the invention such as PfSSB are labelled on a short loop of residues joining the two beta sheets ( ⁇ and ⁇ ').
  • proteins of the invention such as PfSSB are labelled on a short loop of residues (Li-i'), joining two beta sheets ( ⁇ and ⁇ ').
  • proteins of the invention such as PfSSB are labelled on a short loop comprising residues 91 - 96 (L1-1'), joining two beta sheets ( ⁇ and ⁇ ').
  • proteins of the invention such as PfSSB are labelled on a short loop comprising residues 91 - 96 (L1-1'), joining two beta sheets ( ⁇ and ⁇ '), most suitably on amino acid residue 92, 93 or 94.
  • proteins of the invention such as PfSSB are labelled on a short loop comprising residues 91 - 96 (L1-1'), joining two beta sheets ( ⁇ and ⁇ '), most suitably on amino acid residue C93.
  • Residues for labelling are suitably wild type cysteine or are suitably mutated to cysteine as necessary.
  • labelling position 92 of protein of the invention such as PfSSB
  • a G92C mutation is made;
  • labelling position 94 an E94C mutation is made.
  • a protein of the invention has only one cysteine; suitably when sites other than that corresponding to C93 of PfSSB are labelled, C93 is mutated to an amino acid other than cysteine, such as C93A.
  • C93 is mutated to an amino acid other than cysteine, such as C93A.
  • any cysteines present at positions other than C93 - suitably any such cysteine(s) are mutated to residues other than cysteine, such as mutated to alanine, so that the proteins of the invention suitably comprise only one cysteine.
  • Labelling at the corresponding three-dimensional site may be carried out such as at position 151 of protein of the invention such as PfSSB.
  • labelling position 151 a K151C mutation is made.
  • Amino acids adjacent to C93 may be labelled.
  • 'adjacent means adjacent in three dimensional space. This may mean near neighbour residues such as position 92 or position 94 which are both near neighbours of C93 i.e. they are the amino acid before or the amino acid after the one specified.
  • Amino acids close in three-dimensional space to those exemplified for labelling might also be used, such as amino acids close in three-dimensional space to C93.
  • Amino acids close in three-dimensional space to C93 include:
  • PfSSB C93 neighbouring in space locations can be expected to give similar signal if mutated to cysteine and labelled with an environmentally sensitive fluorophore.
  • C93 and neighbouring residues are shown in Figure 21: ssDNA is shown in orange (as a ribbon/ladder); two SSB subunits are shown. When adjacent residues are labelled the expectation is that a similar signal to that achieved with the preferred C93 site is observed.
  • the C93 labelling surface accessibility (ASA) value was over 30%, in higher end of intermediately accessible residues.
  • the immediately adjacent residues to C93 are the G92 and E94, which have ASA values of 35.3% and 55.8%, respectively.
  • K151 on another loop is very close in three dimensional space to the C93 residue and is also expected to give valuable signal when labelled.
  • the ASA value for this residue is 68.8%.
  • the SSB from Vlasm odium falciparum has been characterised and its structure determined 7 9 , which overall is very similar to bacterial SSBs.
  • the crystal structure of P SSB (24.5 kDa) DNA-binding domain showed it be structurally highly similar with the Z?cSSB (19.5 kDa) with major structural differences can be expected to be at the intrinsically disordered region, which is 57 amino acids long in ffcSSB and over 80 amino acids long in P SSB ⁇ 8 ⁇ 10 ⁇ n . This region is unstructured and hence not visible in the crystal structures of the both species SSBs.
  • the 65-70-base binding mode that is 65-70 bases of ssDNA wrap around the SSB tetramer, predominates over the 35-base binding mode.
  • Plasm odium SSB such as PfSSB has been used to develop a probe for ssDNA by forming an adduct with a fluorophore.
  • the best combination of fluorophore and labelling position is believed to be a diethylammoniumcoumarin at C93.
  • This adduct showed tight and rapid binding of ssDNA with a maximum fluorescence change of 20-fold. This fluorescent signal could then be used to characterise both the mode and kinetics of binding.
  • the invention provides a modified Plasm odium SSB comprising at least one detectable label attached to an amino acid of the protein.
  • the detectable label is on a short loop of residues joining the two beta sheets ( ⁇ and ⁇ ').
  • the invention relates to a ssDNA binding molecule, such as a protein, comprising a polypeptide wherein said polypeptide comprises a cysteine residue for attachment of at least one reporter moiety, and comprises at least one reporter moiety attached thereto.
  • said cysteine residue for attachment of at least one reporter moiety is positioned such that said reporter moiety undergoes a change in fluorescence upon ssDNA binding.
  • sensors of the present invention offer signals of at least twelve times, more typically approximately twenty times signal in the bound compared to unbound state. This is a dramatic improvement on the performance of prior art biosensors.
  • the Plasm odium protein has a very similar overall three dimensional structure to the E.coli protein. However, the arrangement of DNA binding to the Plasmodium proteins is very different. One difference is that the DNA binds in the opposite direction to the Plasm odium protein compared to the E.coli protein. This is an extremely surprising observation. It is especially surprising considering the overall three dimensional structural similarity between the two proteins.
  • the inventors have labelled the ssDNA binding protein of Plasm odium at the position of cysteine 93. This is a naturally occurring cysteine present in the wild-type protein. This site is near to the DNA binding groove of the ssDNA binding protein.
  • the sequence location of the dye attachment site is completely different to any of the prior art biosensors.
  • the three dimensional structure of the dye attachment site is completely different to any prior art biosensor.
  • dye attachment to the prior art E.coli ssDNA binding protein based biosensors has been at the very end of a loop present on the protein surface of the E.coli polypeptide.
  • the inventors have attached the dye to an entirely different structural part of the Plasm odium protein. The attachment is not on the same loop as the E.coli prior art sensors.
  • the dye is attached to a surface of the Plasm odium protein, but on a very different secondary structure.
  • the label or dye is attached to the C93 residue of the Plasm odium falciparum single stranded DNA binding protein.
  • the SSB of the invention is modified in order to include a detectable label attached to an amino acid of the protein and whose detectable characteristics alter on binding single stranded DNA.
  • This alteration may, but not necessarily, result from a change in protein conformation. Whether the alteration is due to a change in the protein confirmation or not, the change in the detectable characteristics is due to an alteration in the environment of the label, which is bound to the single stranded DNA binding protein.
  • Labels used with the invention can give various signals, but preferred labels are luminescent labels. Luminescent labels include both fluorescent and phosphorescent labels. However, the use of other labels is envisaged. For example, electrochemical labels could be used wherein the alteration in the environment of the labels will give rise to a change in redox state. Such a change may be detected using an electrode.
  • the detectable label is preferably a fluorescent label. The use of fluorescent labels, which may be excited to fluoresce by exposure to certain wavelengths of light, is preferred.
  • Attachment of the detectable label to the SSB may sometimes reduce the affinity of the protein for single stranded DNA. However, this does not prevent an alteration of the detectable characteristics on binding single stranded DNA which can be quantitatively measured.
  • the fluorescent label is a coumarin or a rhodamine.
  • labels of the invention may have shorter excitation wavelengths of between about 400-46onm or longer excitation wavelengths of between about 540-6oonm.
  • Rhodamine dyes to the Plasm odium ssDNA binding protein, for example at cysteine 93. This produces a signal change, but the sensor requires careful handling in titration type experiments. The reason is that Rhodamine type dyes have tended to produce nonlinear signal changes when attached at C93. Thus, in some embodiments it is possible to use Rhodamine type dyes. However, Rhodamine type dyes attached to Plasm odium proteins at C93 may produce complex responses requiring careful interpretation. For these reasons, Coumarin type dyes are preferred. Coumarin type dyes are especially preferred for attachment at C93 of the Plasm odium ssDNA binding protein.
  • the dyes/labels used in the invention are Coumarin type dyes/labels.
  • any coumarin may be used, including the iodoacetamide- and maleimide- linked diethylaminocoumarins.
  • diethylaminocoumarins include N-[2-(i- maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide and N-[2- (iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide (sometimes referred to as referred to as 7-diethylamino-3-((((2- iodoacetomido)ethyl)amino)carbonyl)coumarin) (IDCC).
  • the coumarin used is IDCC.
  • fluorophore types known to have fluorescence intensity that depends on physical environment such as interactions with protein surfaces, may be used including:
  • MIANS (2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid)
  • IAEDANS (5-[2-[(2-Iodo-i-oxoethyl)amino]ethylamino]-i-naphthalenesulfonic acid) Alexa Fluor 488 maleimide
  • Cy3-maleimide (i-(6- ⁇ [2-(2,5-dioxo-2,5-dihydro-iH-pyrrol-i-yl)ethyl]amino ⁇ -6- oxohexyl)-2-[(iE,3E)-3-(i-ethyl-3,3-dimethyl-5-sulfo-i,3-dihydro-2H-indol-2- ylidene)prop-i-enyl]-3,3-dimethyl-3H-indolium)
  • the SSBs of the present invention have labels attached to certain amino acid(s).
  • the present invention also provides a method for making a modified SSB which involves modification of a SSB to include a detectable label attached to an amino acid of the protein.
  • the label may be attached to the SSB by any conventional means known in the art.
  • the label may be attached via amines or carboxyl residues on the protein.
  • linkage via thiol groups on cysteine residues is especially preferred.
  • the modified SSB may comprise more than one detectable label.
  • the labels are preferably attached to separate amino acid residues.
  • each monomer of the SSB has the same number of labels attached to it. There may be one label per monomer or alternatively two, three or four labels attached to each monomer. Preferably there is one label per monomer. In a further aspect of the invention, where more than one monomer of the SSB tetramer is modified to include a detectable label, such labels may stack.
  • cysteine residues in the sequence of the SSB may be used for the attachment of the label.
  • cysteine residues may be engineered into the sequence of the SSB, suitably by site-directed mutagenesis.
  • the invention provides a SSB wherein a wild-type non-cysteine residue is replaced by a cysteine residue.
  • Site-directed mutagenesis will be performed by methods well known in the art for this purpose. Briefly, however, the gene encoding the SSB is isolated, and oligonucleotide probes are constructed to alter by recombination, or more suitably by amplification, the codon encoding the amino acid which it is desired to change into a codon encoding cysteine.
  • the mutated gene is subsequently expressed, typically in a bacterial expression system, to produce the mutated protein.
  • the invention also provides a SSB wherein the label is attached to a region of the protein surface. Regions of the protein such as a subunit-subunit interaction surface are not exposed and are thus unsuitable for labelling purposes. Surface located residues are more easily accessible for labelling purposes and are less likely to disrupt the overall shape of the protein when labelled.
  • residues chosen for label attachment are located in a region of the protein surface, which is above a binding channel and on loops of the protein structure.
  • 3D structures of several SSBs are known in the art.
  • the residues chosen are not in a region of the protein which would directly interact with the DNA binding surface per se but are close enough to the DNA surface such that the attached label might be affected by the presence of the DNA.
  • This strategy is a compromise between disrupting DNA binding by, for example, sterically hindering DNA binding (by being “too close”) and getting no signal change (by being “too far away”).
  • an alteration in the environment of the label may result from a conformational change in a region of the protein to which the label is not directly bound.
  • Fluorophores will rarely be attached to an amino acid directly, but will instead be attached via a linker.
  • the choice of linker can also have an effect on the way the labelled SSB functions, as the size, shape and flexibility of the linker can change the ability of a linker to come into proximity with other groups.
  • Labels are preferably attached to the SSB in a manner that does not introduce a new chiral centre.
  • the label-protein adduct does not exist in diastereoisomeric form thus allowing a substantially homogenous labelled SSB to be obtained.
  • linkers such as the haloacetamides (preferably iodoacetamides).
  • labelled protein After attachment of the label, labelled protein will usually be purified to separate it from free label and from any mis-labelled protein.
  • the mis-labelled protein may be unlabelled protein with which label did not react or protein where label has attached in the wrong position (either in place of or in addition to the desired label).
  • treatment with a thiol reagent may be included, such as ⁇ -mercaptoethanol, dithiothreitol or sodium 2-mercaptoethanesulfonate as this can improve the fluorescence response of the protein.
  • a homogenous form e.g. pure single-labelled species, may be purified (for example by ion exchange and/or hydrophobic interaction chromatography) to obtain homogenous, double-labelled species.
  • Single and double labelled SSBs can be distinguished by methods such as electrospray mass spectrometry.
  • the detectable label preferably shows an increase in its detectable characteristics upon binding single stranded DNA.
  • this is at least 12-fold, more preferably 20-fold.
  • NUCLEOTIDE BINDING Prior art biosensors such as the E.coli based ssDNA sensor have tended to bind 35 nucleotide segments. The inventors have observed that the 35 nucleotide long ssDNA is insufficient to wrap completely around tetramer. Thus, when binding a 35 nucleotide ssDNA, at most only approximately 2 out of the 4 available fluorescent dye molecules are likely to be affected by binding. This contributes to a lower signal which is a problem of the prior art sensors.
  • nucleotides may wrap so that they only affect 1 of the 4 possible fluorophores - with one end of the ssDNA associating with a protein a certain distance from a first fluorescent dye, continuing around the ssDNA binding protein and affecting one such fluorophore, and ending before reaching the fluorophore located on the third such molecule of the tetrameric sensor, which would produce an even lower signal.
  • the homo-tetramers of the present invention bind in a 65-70 nucleotide mode, which means that each of the tetramers is likely to be affected by ssDNA binding leading to an advantageously larger signal.
  • the Plasm odium ssDNA binding protein has a large "unstructured region" exceeding 80 amino acids in length. By contrast, the E.coli protein has a much shorter 57 amino acid long unstructured region. Without wishing to be bound by theory, the inventors believe that differences in the unstructured region lead to a decreased cooperativity in the Plasm odium protein. It is preferred that the unstructured region is retained in the biosensor molecules of the invention, since deletion of the unstructured region may affect DNA binding. It is an advantage of the invention that certain assay conditions are enabled in which prior art sensors such as E.coli sensor do not work. One example of such conditions is low salt conditions. Prior art E.coli based sensors tend to give peculiar shaped binding curves when used in low salt conditions. By contrast, Plasm odium based sensors of the invention give good results even in low salt conditions. Suitably "low salt” conditions means 20mM.
  • DNA/Sensor ratios are less likely to adversely affect signal.
  • the sensor of the invention performs well with DNA/sensor ratios in the range sensor to DNA (take in 65 base units): From 2 to > 20 fold.
  • the single strand DNA-binding domain protein family is an example of a family of proteins that interact with single stranded DNA and unwound double stranded DNA.
  • the SSB family of proteins play essential roles in DNA replication, recombination and repair both in prokaryotes and eukaiyotes.
  • Members of this family of proteins have been investigated from several organisms and there are structures of the protein in the presence and absence of DNA.
  • the invention may be applied to any Plasm odium single stranded DNA binding protein.
  • the invention is applied to the Plasm odium falciparum single stranded DNA binding protein.
  • the invention is applied to a polypeptide derived from, having or consisting of the sequence of SEQ ID NO:i
  • Plasm odium ssDNA binding protein is used.
  • the N-terminus of the protein comprises the DNA binding domain (DBD).
  • DBD DNA binding domain
  • the N-terminus of the protein is not truncated.
  • the protein contains the whole N-terminal region of the protein.
  • the C terminal end of the Plasm odium ssDNA binding protein may be truncated.
  • up to 120 amino acids may be removed from the C terminal end of the polypeptide, with the resulting truncated protein expressing and purifying adequately.
  • the invention also includes sensor molecules as described herein with up to 120 amino acids truncated from the C terminal end; suitably 100 or fewer amino acids are truncated.
  • the final ⁇ 87 residues are not likely to be involved in DNA binding. Therefore suitably 87 or fewer amino acids are truncated. If 87 residues are removed, then the protein may be less stable and may not be suitable when high stability is desired.
  • suitably 80 or fewer amino acids are truncated; suitably 60 or fewer amino acids are truncated.
  • the apicoplast localisation sequence is suitably not part of the sensor polypeptide of the invention.
  • the ALS is 76 amino acids long at the N- terminus of the naturally occurring sequence.
  • the polypeptide of the invention is expressed without the 76 amino acid ALS.
  • the first (77 th ) amino acid of Plasm odium protein without the ALS is methionine.
  • this initiator methionine is cleaved from the mature protein.
  • the first amino acid of the biosensor of the invention is the 78 th amino acid of the full wild-type sequence (i.e. the wild-type sequence minus the ALS and minus the first methionine at position 77).
  • the amino acid numbering should be treated as corresponding to the equivalent section of the full length Plasm odium falciparum single stranded DNA binding protein reference sequence and not as an 'absolute' or rigidly inflexible numeric address.
  • the address given will still be C93 (rather than e.g. C16) - this will be easily understood by the skilled reader to refer to the amino acid of the corresponding context with reference to the Plasm odium falciparum single stranded DNA binding protein sequence of SEQ ID NO:i, as is conventional in the art.
  • the exemplary Plasm odium falciparum single stranded DNA binding protein has been studied in detail and each residue has been classified as set out in Table A below. Table A lists all residues, based on surface exposure.
  • the PDB file used for the analysis was the 3ULP that was modified by removing the ssDNA.
  • the structure does not include the ALS sequence or the unstructured C-terminal cooperativity domain.
  • the classification is as follows, making use of information on surface exposure:
  • Buried Residues in the core of the structure where mutations are likely to affect the function of the sensor.
  • the relative accessible surface area (ASA) of a residue is its ASA calculated using DSSP (Kabsch, W. and C. Sander (1983). "Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features.” Biopolymers 22(12): 2577-637.) divided by its nominal maximum area as defined by (Chothia, C. (1976). "The nature of the accessible and buried surfaces in proteins.” J Mol Biol 105(1): 1-12).
  • a residue is defined as exposed if its relative ASA is at least 40% of its nominal maximum area.
  • a residue is defined as buried if its relative ASA is less than 10% of its nominal maximum area.
  • residues in the polypeptide part of a sensor molecule of the invention have at least 90% sequence identity to SEQ ID NO:i. Suitably any differences are conservative substitutions.
  • residues in the polypeptide part of a sensor molecule of the invention have 100% similarity to SEQ ID NO:i. More suitably residues shown as 'buried' in table A are not mutated. Thus suitably residues in the polypeptide part of a sensor molecule of the invention which correspond to residues shown as 'buried' in table A are not mutated relative to SEQ ID NO:i.
  • residues in the polypeptide part of a sensor molecule of the invention shown as 'buried' in table A comprise the same residue as at the corresponding position in SEQ ID NO:i.
  • the polypeptide component of the sensor molecule of the invention suitably comprises amino acid sequence having 100% sequence identity to those residues shown as 'buried' in table A.
  • residues shown as 'intermediate' in table A may be mutated.
  • residues in the polypeptide part of a sensor molecule of the invention which correspond to residues shown as 'intermediate' in table A may be mutated relative to SEQ ID NO:i.
  • residues in the polypeptide part of a sensor molecule of the invention shown as 'intermediate' in table A may comprise a different residue (or no residue) from the corresponding position in SEQ ID NO:i.
  • a biosensor of the invention has at least 60% sequence identity to SEQ ID NO: 1.
  • the polypeptide component of the sensor of the invention suitably comprises amino acid sequence having at least 60% sequence identity to those residues shown as 'intermediate' in table A.
  • the polypeptide component of the sensor of the invention suitably comprises amino acid sequence having at least 68% sequence identity to those residues shown as 'intermediate' in table A, suitably least 70% sequence identity, suitably least 74% sequence identity, suitably least 78% sequence identity, suitably least 82% sequence identity, suitably least 86% sequence identity, suitably least 90% sequence identity, suitably least 94% sequence identity, suitably least 98% sequence identity to those residues shown as 'intermediate' in table A.
  • these 'intermediate' residues are in fact partially buried.
  • these 'intermediate' residues are only mutated by substitution with a conservative residue relative to SEQ ID NO: 1.
  • the non- identical residues noted above comprise only conservative substitutions relative to the corresponding residue in SEQ ID NO: 1.
  • sequence similarity takes account of sequence identity and also takes account of conservative substitutions (i.e. non-identical residues but where the residue is similar or conserved compared to the original residue). Assessing sequence similarity is well known in the art. Examples are provided in the examples section. In case any further guidance is needed, suitably the following parameters are used in the algorithm for calculating sequence similarity: BLOSUM62 matrix, gap penalty 10.0, gapextend penalty 0.5; more suitably BLOSUM62 matrix, gapopen 10.0, gapextend 0.5, endopen 10.0, endextend 0.5, pairwise alignment.
  • polypeptide component of the sensor of the invention has at least 50% sequence similarity to SEQ ID NO: 1, suitably at least 60% sequence similarity, suitably at least 63% sequence similarity, suitably at least 65% sequence similarity, suitably at least 70% sequence similarity, suitably at least 75% sequence similarity, suitably at least 80% sequence similarity, suitably at least 85% sequence similarity, suitably at least 90% sequence similarity, suitably at least 95% sequence similarity, suitably at least 98% sequence similarity, suitably at least 99% sequence similarity, most suitably 100% similarity to SEQ ID NO: 1.
  • residues shown as 'intermediate' in table A are not mutated.
  • residues in the polypeptide part of a sensor of the invention which correspond to residues shown as 'intermediate' in table A are not mutated relative to SEQ ID NO:i.
  • residues in the polypeptide part of a sensor of the invention shown as 'intermediate' in table A comprise the same residue as at the corresponding position in SEQ ID NO:i.
  • sequence similarity or sequence homology (identity) scores are calculated for the section or segment of the polypeptide/polynucleotide which corresponds to or aligns with the reference sequence such as SEQ ID NO: l. Therefore, if the target sequence is shorter than the reference sequence such as SEQ ID NO: ⁇ (e.g. a truncation) then the score is calculated only for the length of the shorter or truncated sequence, i.e. for the 'overlapping region' between the two sequences.
  • residues shown as 'exposed' in table A may be mutated.
  • residues in the polypeptide part of a sensor of the invention which correspond to residues shown as 'exposed' in table A may be mutated relative to SEQ ID NO:i.
  • residues in the polypeptide part of a sensor of the invention shown as 'exposed' in table A may comprise a different residue (or no residue) from the corresponding position in SEQ ID NO:i.
  • the polypeptide component of the sensor of the invention suitably comprises amino acid sequence having at least 21% sequence identity to those residues shown as 'exposed' in table A, suitably at least 30% sequence identity, suitably at least 40% sequence identity, suitably at least 50% sequence identity, suitably at least 60% sequence identity, suitably at least 63% sequence identity, suitably at least 65% sequence identity, suitably at least 70% sequence identity, suitably at least 75% sequence identity, suitably at least 80% sequence identity, suitably at least 85% sequence identity, suitably at least 90% sequence identity, suitably at least 95% sequence identity, suitably at least 98% sequence identity, suitably at least 99% sequence identity, most suitably 100% identity to those residues shown as 'exposed' in table A.
  • Plasmodium falciparum SSB Other SSBs, in particular other Plasmodium SSBs, may be equally used. Exemplary sequences are presented below. Sequences are given for a variety of Plasmodium species with sequence alignment after. This illustrates the close sequence homology to P. falciparum protein, the exemplary sensor protein, in the DNA-binding region:
  • B3L6I1 B3L6I1_PLARH TEVVLSYNRGDL IFLDDRRNFISRNTSNVQSTESASSSNEANIANSL GGNADE A0A0D9QP36
  • A0A0D9QP36_PLAFR TEVVLSYNRGDL IFLDDRRNFISRNASNAQSSESSTSTSEANVSSSL GGNSDE W7A536 W7A536_9APIC TEVVLSYNRGDL IFLDDRRNFISRNVSNVQSSESTSSNSEANVASSL GGNSDE A5RB16 A5RB16_PLAVS TEVVLSYNRGDL IFLDDRRNFISRNASNVQSSESPPSTSEANVANSL GGNSDE R6URM8 R6URM8_9APIC TEVVLSYNRGDL IFLDDRRNFISRNASNVQSSESPSSTSEPSVANSL GGNSDE W7RL30 W7RL30_PLAFO TEIILSYNRGDL IFLDDRRNFN
  • the homo-tetramers into which the sensor molecules associate are formed by binding of 2 dimer pairs to one another. It is possible to introduce mutation(s) to reduce the interaction of the pairs of dimers. For example, a Y156 mutation such as Y156R may be made to reduce the association of dimers into tetramers, thereby promoting dimer formation.
  • the invention relates to ssDNA sensors comprised of dimers of two individual sensor polypeptides of the invention.
  • the SSB structure indicates weaker interactions between pairs of subunits: the inventors teach that disruption of this dimer-dimer interface leads to viable dimeric proteins.
  • Z?cSSB changing tyrosine 156 into arginine in the equivalent location, .ECSSB-Y78R has been shown to lead to stable dimer of Z?cSSB (Landwehr, M., Curth, U., and Urbanke, C. (2002) A dimeric mutant of the homotetrameric single-stranded DNA binding protein from Escherichia coli, Biol Chem 383, 1325-1333). Kozlov, A.
  • This mutation has been made in PfSSB and the dimer has been labelled with IDCC on the C93 location.
  • This molecule is stable and has a 19-fold signal change when binding to ssDNA and can be used as a dimeric form PfSSB-based ssDNA biosensor.
  • the location of Y156 residues on each subunit is shown in Figure 21.
  • the sensor of the invention has one or more mutations from Table SB; suitably the sensor of the invention has two or more mutations from Table SB;
  • the sensor of the invention has three or more mutations from Table SB; suitably the sensor of the invention has all four mutations from Table SB.
  • Aspartate, D189 has two lysine residues in close proximity, which are K84 and K187. Either one of the lysines is close enough for possible salt bridge formation.
  • the pair K84 and D189 is the most likely as they do fall closer to each other on the crystal structure. Suitable mutation to disrupt the dimer-dimer interaction would be to oppose the charges individually and this would break the salt bridge and lead destabilisation of the dimer-dimer interface (K84D, K187D and D189K).
  • the invention relates to a tetramer, each tetramer comprising 4 individual polypeptides of the invention.
  • TAGS It is often useful to tag proteins of the invention, for example to facilitate their purification after recombinant production.
  • Suitably tags may be placed at the extreme C-terminus or the extreme N-terminus of the sensor molecule.
  • tags are placed at the C-terminus of the protein. Most suitably the N- terminus of the protein is not tagged.
  • one tag per protein molecule is used.
  • Multiple tags per protein molecule may be used if desired, including multiple copies of the same tag or two or more different tags, for example it may be desirable to use a 6 His-tag for purification and in addition to use a Myc-tag for detection.
  • Tags may be removed from the sensor protein, for example by proteolytic cleavage, or may be retained on the sensor protein during use.
  • a hexahistidine tag (6his) may be added to the polypeptide part of the sensor molecule of the invention to simplify purification; most suitably a C-terminal hexahistidine tag is used. 6 His is a particularly useful tag for purification on nickel substrates. However, any suitable tag known in the art may be used. Alternatively, the sensor molecule of the invention may be tagless. Tagless purification (if needed) is well known in the art.
  • polypeptide components of the sensor molecules of the invention may be produced by standard recombinant techniques, such as creating a nucleic acid encoding the amino acid sequence of the polypeptide, and then expressing the polypeptide in a host such as E.coli. Alternatively an in vitro translation may be used. Alternatively the polypeptide itself may be chemically synthesised.
  • the polypeptide(s) may be purified by any suitable method known in the art, such as 6His tagging the protein then purification using Ni-NTA beads.
  • any suitable technique may be used such as site directed mutagenesis.
  • mutant PCR primers or oligonucleotides containing the desired nucleotide sequence may be annealed to a template and ligated, extended or amplified to produce a mutated nucleotide sequence encoding the desired substitution.
  • the desired nucleotide sequence may be synthesised chemically.
  • the sensors of the invention have the advantage of being usable under a wide range of pH conditions.
  • the pH of the assay is in the range 6.0 to 9.0. More suitably the pH of the assay is in the range about 6.5 to about 8.5. Most suitably the pH of the assay is in the range 6.5 to 8.5.
  • salt conditions for assays or use of the sensors of the invention are from 20 mM to >200 mM.
  • Preferred salts are those mentioned in the examples section.
  • the labelled SSBs of the invention can be used in assays for general biochemical use for detecting single stranded DNA in a sample. For example, in detecting strand separation of double stranded DNA which is catalysed by helicases and allows further processing such as repair and replication.
  • the labelled SSBs of the invention can be used in assays to measure the removal of single stranded DNA, or in assays to measure the decrease in double stranded DNA.
  • These assays can be qualitative or quantitative.
  • the invention is particularly useful for following the kinetics of reactions, because of the rapid reaction time of the SSBs.
  • the assays can be used in screening methods for inhibitors of DNA processing enzymes. Such assays comprise assaying single stranded DNA levels in vitro using a SSB according to the present invention in the presence and absence of the inhibitors and assaying for an alteration in the single stranded DNA levels.
  • the single stranded DNA may be a single strand of DNA or may be a single stranded region of a DNA duplex.
  • the sample may be from any source, including serum, urine, saliva, sweat, tissue culture, cell extracts, cell lines, food, beverages, pharmaceuticals and environmental (for example water). If concentrations of single stranded DNA in the sample are high, samples may be diluted as necessary to achieve accurate quantification of single stranded DNA levels.
  • the kinetics of these methods may depend on salt concentration or the presence of a particular anion.
  • a skilled person would be able to select the appropriate conditions to carry out the methods of the invention.
  • the sensors of the invention are advantageously able to perform in a wide range of salt conditions, including low salt conditions, which is an advantage over prior art sensors.
  • the invention provides a method for detecting single stranded DNA in a sample comprising the steps of:
  • the change detected in (ii) can be related to the concentration of single stranded DNA in the sample.
  • the invention also provides a modified SSB of the invention, for use in an assay of single stranded DNA.
  • a further aspect of the invention provides a polypeptide sequence of SEQ ID NO: l in which one or more wild type amino acid residues are changed to a cysteine residue.
  • a further aspect of the invention provides a polypeptide sequence of SEQ ID NO: l in which the wild type cysteine residue has a label attached.
  • the invention further provides PfSSB polypeptide sequence as disclosed herein with a label attached, suitably at C93 or the amino acid residue corresponding to same.
  • the invention further provides the nucleotide sequences which encode the polypeptide sequences disclosed herein.
  • composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
  • the invention in another aspect, relates to a process for making a SSB as described above which method comprises modification of a SSB to include a detectable label attached to an amino acid of the protein.
  • the invention relates to a fluorescent reagentless biosensor for single-stranded DNA using Plasm odium SSB.
  • the invention relates to a fluorescent biosensor for single-stranded DNA using Plasm odium such as Plasm odium falciparum single stranded DNA binding protein as the polypeptide, or protein scaffold.
  • the sensors of the invention are structurally different from the SSB proteins as they occur in nature, due to specific mutational differences compared to the naturally occurring sequences and due also to the presence of dye molecules attached to the polypeptides which are not present in nature. Therefore the invention clearly involves the 'hand of man' and is eligible for patent protection, presenting molecules and methods which do not occur in the natural world.
  • Figure 1 shows fluorescent spectra and titrations of DCC-PfSSB with ssDNA.
  • Figure 2 shows effect of ionic strength on PfDCC-SSBC93 binding to ssDNA.
  • the titrations were performed at same conditions as in Figure lB.
  • the solutions contained 250 nM PfDCC-SSBC93 in Graph A and C and 200 nM in Graph B.
  • A) The breakpoint of fluorescence increase is 530 nM of dT35 at high salt and 544 nM at low salt concentration.
  • B) The breakpoint for fluorescence increase for dT70 is 187 nM of dT70 at high salt and 193 nM dT70 at low salt.
  • C) The breakpoint for fluorescence increase for polydT was at 266 nM at high salt and 269 nM at low salt concentration of polydT.
  • the data was fitted to double exponential, where the first phase rate constant followed concentration dependence.
  • the second phase was significantly smaller in amplitude for all ssDNA lengths and less than 20% of the entire signal at all ssDNA concentrations. Only second slow phase fitted for dT70 followed concentration dependence.
  • B) Association kinetics at low ionic strength conditions (B) fast phase and (C) slow phase. At low ionic strength the traces fitted well double exponentials and the observed rate constants for both phases followed concentrations dependence.
  • the second order rate constants determined from the linear fits are given in Table 3. The measurements were done on the stopped-flow apparatus and the final concentration of the DCC-PfSSBC93 was 5 nM and the ssDNA was varied
  • Figure 4 shows DCC-PfSSBC93 association kinetics to various lengths of ssDNA when the DCC-PfSSBC93 in excess at high ionic strength.
  • Figure 5 shows association kinetics with excess DCC-PfSSB over ssDNA at high ionic strength.
  • Figure 6 shows dissociation kinetics at high ionic strength.
  • the measurement were done by premixing 20 nM DCC-PfSSBC93, 25 nM dT70/50 nM dT35/25 nM polydT to form the PfSSB-ssDNA complex before mixing with varying concentrations of wtSSB.
  • the final concentrations in the reaction were 10 nM DCC- PfSSBC93, 12.5 nM dT70/ 25 nM dT35/ 12.5 nM polydT and 0.25 - 4 ⁇ wtSSB.
  • the measurements were repeated at high (200 mM NaCl) and low (20 mM NaCl) ionic strength buffers.
  • Figure 7 shows dissociation kinetics at low ionic strength.
  • Figure 8 shows dependence of dissociation kinetics on the wtSSB concentration.
  • the measurements (A)-(C) have been measured at high ionic strength (200 mM NaCl) and the measurements (D)-(F) have been done at low ionic strength (20 mM NaCl).
  • A dT35, high salt.
  • B dT70, high salt.
  • C polydT, high salt.
  • D dT35, low salt.
  • E dT70, low salt.
  • the values for gradients for all linear fits in graphs (A) to (E) are given in Table 5.
  • the average koff of four concentrations of wtSSB measured is 1.3 s-i for the fast phase and 0.12 s-i for the slow phase.
  • Figure 9 shows plasmid unwinding by PcrA helicase.
  • Figure 10 shows AddAB helicase assay with linear dsDNA.
  • Figure 11 shows Plasmodium falciparum ssDNA bp; DNA is shown in brown (see the strand wound around the protein structure of PfSSB). The labelling position is highlighted only on one of the four subunits in the structure of PfSSB (C93 in magenta - see amino acid on central lower region of PfSSB).
  • Figure 12 shows Escherichia coli ssDNA bp; DNA is shown in blue (see the strand wound around the protein structure of EcSSB). The labelling position is highlighted only on one of the four subunits in the structure of EcSSB (G26 in red - see amino acid on far right lower loop of EcSSB).
  • Figure 13 shows a graph
  • Figure 14 shows a graph
  • Figure 15 shows a graph
  • Figure 16 shows a graph
  • Figure 17 shows a graph.
  • Figure 18 shows a graph.
  • Figure 19 shows a diagram.
  • Figure 20 shows a graph
  • Figure 21 shows a diagram
  • Figure 22 shows a diagram
  • the four subunits are coloured (shaded) differently and DNA is shown in brown (PfSSB) and blue (EcSSB).
  • PfSSB brown
  • EcSSB blue
  • the labelling position is highlighted only on one of the four subunits in each structure for illustration purposes - suitably each subunit is labelled.
  • PfSSB C93 in magenta
  • EcSSB G26 in red).
  • C93 of PfSSB is located on a short loop of residues 91 - 96 (L1-2), joining two beta sheets ( ⁇ and ⁇ 2).
  • G26C of EcSSB is located on the end of a hairpin loop, residues 21 -29 (L1-1'), joining two interacting beta sheets ( ⁇ and ⁇ ').
  • Plasm odium proteins according to the invention show significant and demonstrable advantages which are not possessed by the prior art proteins.
  • EcSSB E. coli protein
  • PfSSB Plasm odium SSB
  • Non-linear response is demonstrated with prior art DCC-EcSSB at low salt (20 mM NaCl), titrating dT70 as DNA into ⁇ 250 nM biosensor.
  • Fig 15 shows prior art DCC- EcSSB
  • Fig 16 shows an example of the invention DCC-PfSSB. The improved response using the invention is clearly apparent.
  • DCC-EcSSB to dT70 at high concentrations. This is due to slow transition to 35- base-mode binding giving lower fluorescence with prior art DCC-EcSSB.
  • DCC-PfSSB an example of the invention DCC-PfSSB.
  • Fig 17 shows prior art DCC-EcSSB
  • Fig 18 shows an example of the invention DCC- PfSSB.
  • the assay measures double-stranded DNA (dsDNA) unwinding by the helicase AddAB.
  • the diagram in Figure 19 illustrates the basis of the assay.
  • the reaction in the presence of prior art DCC-EcSSB or an example of the invention DCC-PfSSB (at identical concentrations) and at low ionic strength (20 mM NaCl), was initiated by addition of ATP.
  • ATP-coupled unwinding leads to an increase of ssDNA to which DCC-SSB binds. This was observed as an increase in fluorescence. Once the entire dsDNA is unwound, a steady state of fluorescence should be observed.
  • PfSSB wild-type and single cysteine mutants were expressed from pET22b in C41(DE3) E. coli cells using the T7 promotor system (Lucigen, WI, U.S. A).
  • the cells were grown at 37 °C in 0.5 L x 4 LB cultures to OD 595 of 0.6 and the expression induced using 1 mM IPTG.
  • the cells were harvested after 5 h by centrifugation and resuspended in 80 ml 50 mM Tris HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 10% sucrose, and stored -80 °C.
  • PfSSB was purified as described for E. coli SSB 2 with minor modification and omitting the polymine P precipitation step. The purification was done at 0-4 °C.
  • the cells were thawed and 1 protease inhibitor tablet (Roche) per 50 ml of cells was added. Cells were lysed using probe sonication, 4 x 20 s. Lysate was spun for 20 min at 38000 g. Solid ammonium sulphate was gradually add to 150 g/L and stirred for 30 min. The solution was spun at 14 000 g for 20 min and the pellet was resuspended with 50 mM Tris HCl (pH 8.3), 200 mM NaCl, 1 mM EDTA and 20% glycerol (v/v) and stirred for 30 min to improve the resuspension of PfSSB.
  • 1 protease inhibitor tablet (Roche) per 50 ml of cells was added. Cells were lysed using probe sonication, 4 x 20 s. Lysate was spun for 20 min at 38000 g. Solid ammonium sulphate was gradually add to 150 g/L and stirred for 30 min
  • the solution was spun for 20 min at 38 000 g to remove any impurities and passed through 0.2 ⁇ syringe filter membrane before loading on to a 5 ml heparin column (GE healthcare, Little Chalfont, U.K.).
  • the column was pre-equilibrated with buffer A consisting of 50 mM Tris HCl (pH 8.3), 1 mM EDTA, 1 mM DTT and 20% glycerol (v/v).
  • the sample was loaded by mixing with the buffer A at ratio of 16/84 for sample/buffer A immediately before loading to column to prevent SSB precipitation known to take place at low salt conditions.
  • SSB was dialysed in a SnakeSkin Pleated Dialysis Tubing (Life technologies, Thermoscientific, Glasgow, U.K.) ) to storage buffer of 50 mM Tris HCl (pH 8.3), 1 mM EDTA, 500 mM NaCl, 1 mM DTT and 50% glycerol (v/v) overnight and concentrated using Viva Spin concentrator with a molecular weight cut-off of 10 000 Da. The concentration of SSB was determined using absorbance at 280 nm with molar extinction coefficient of 95 800 M "1 cm "1 for the tetramer. SSB was stored at -80 °C.
  • pCERoriD plasmids for PcrA helicase assay were prepared as prescribed before 4 .
  • the linear dsDNA was prepared by digestion of p EKoriD plasmids with EcoRI restriction endonuclease (Roche) and gel purified from 1 % agarose gels using QIAGEN gel extraction kit (QIAGEN Ltd, Manchester, U.K.).
  • dT 35 , dT 70 and polydT and other materials unless otherwise stated were obtained from Sigma-Aldrich (Gillingham, U.K.).
  • IDCC N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide
  • Fluorophore labelling of PfSSB Typically, 10 mg of SSB wild-type and its cysteine mutants were labelled using iodoacetamide-thiol chemistry and all labelling steps were performed at room temperature.
  • the PfSSB was incubated with 10-fold excess of DTT over PfSSB monomers for 20 min. DTT was removed by elution through PD-10 column with the labelling buffer (20 mM Tris-HCl pH 7.5, 1.0 mM EDTA, 500 mM NaCl, 20% (v/v) glycerol). IDCC was added in 2.5-fold excess over ⁇ 25 ⁇ PfSSB monomers and incubated 2 hours on end-to-end rotor.
  • IDCC was removed from the reaction using 2-mercaptoethanol sulphate by adding it in 10-fold excess over monomers and incubating for 20 min.
  • the incubation mixture was passed through 0.2 ⁇ syringe filter membrane (Pall Life Sciences, Portsmouth, U.K.) before loading on to a 10 ml P4 gel filtration column (Biorad) pre-equilibrated with the labelling buffer.
  • Labelled-PfSSB was isolated by elution with labelling buffer and if required concentrated using Viva spin concentration with a molecular weight cut-off of 10 000 Da.
  • the concentration of IDCC-labelled PfSSB was determined from the 430 nm absorbance using extinction coefficient for the IDCC, which 44 800 M "1 cm "1 at that wavelength.
  • Absorbance and Fluorescence Measurements Absorbance measurements were done on a Jasco spectrophotometer (Jasco Analytical Instruments Inc., Easton, MD, U.S.A.) using 3 mm pathlength quartz cells at room temperature. Fluorescence measurements were taken on Cary Eclipse spectrofluorometer (Varian, Palo Alto, CA, U.S.A.) with a xenon lamp using 3 mm pathlength quartz cells at room temperature. The fluorescence spectra were measured at the excitation and emission wavelengths given in Table 1, determined from spectra. All fluorescent property and kinetic measurements were performed at 20 °C.
  • the buffer had 25 mM Tris-HCl (pH 7.5), 200 mM NaCl and 10 ⁇ BSA. At low ionic strength buffer NaCl concentration was lowered to 20 mM.
  • Fluorescence titrations were done by excitation at 433 nm and emission at 473 nm for all ssDNA lengths.
  • AFluorescence (F ⁇ - min )(-b+sqrt(b 2 +4(K- 1 )PL))/(2L(K- 1 ))).
  • b KX + P + L -XP
  • X [unlabelled PfSSB]
  • L [DCC-PfSSB]
  • P [total ssDNA]
  • K ratio of K d values for the two SSB species
  • F min fluorescence in the absence of DNA
  • max fluorescence of DCC-PfSSB ssDNA. Stopped-flow fluorescence.
  • Plasmid unwinding assay using PcrA helicase Plasmid unwinding was measured on a stopped flow apparatus at 30 °C in buffer containing 50 mM Tris HCl pH 7.5, 100 mM KC1, 1 mM EDTA, lOmM MgCl 2 and 10% (v/v) ethanediol.
  • a typical reaction contained 0.5 nM pCERoriD plasmid, 190 nM PcrA, 2 nM RepD, 200 nM DCC-SSB or DCC-PfSSB and 1 mM ATP.
  • the plasmid was incubated with RepD for 30 s to allow RepD nicking.
  • the SSB was added to this syringes, A and B.
  • the ATP was added to syringe B and the reaction was mixed.
  • IDCC N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide) 6-IATR, 6-iodoacetamidotetramethylrhodamine
  • Native Plasmodium falciparum SSB is transcribed and translated from nuclear DNA with a 76 amino acid apicoplast localization sequence (ALS) that is cleaved off once delivered to apicoplast.
  • ALS apicoplast localization sequence
  • the PfSSB was expressed without the localization sequence, but amino acids are numbered according the entire translated amino acid sequence.
  • PfSSB has a single cysteine C93 that is located on the surface of PfSSB and was expected to be accessible to labelling using iodoacetamide/maleimide chemistry.
  • Excitation and emission spectra were measure with 250 nM DCC-PfSSB in the presence and absence of 595 nM ssDNA at high salt.
  • the polydT concentration was calculated as concentration of 70-base binding sites. Average length of polydT is 500 bases.
  • the signal change is given as a ratio of fluorescence with nucleic acid divided by fluorescence without nucleic acid at wavelength of maximum emission.
  • DCC-PfSSB fully labeled at C93 with IDCC (named hereafter DCC-PfSSB), overall gave the best characteristics and these are now described in detail.
  • DCC-PfSSB The titration of DCC-PfSSB with dT 70 gave linear increase in fluorescence, followed by a sharp break to a constant level ( Figure IB). This indicates that the binding of DCC-PfSSB is tight and stoichiometric with one DCC-PfSSB binding to one dT 70 .
  • the fluorescence increase with dT 35 titration is also linear but shows two phases with different amplitudes, before the breakpoint.
  • Second order binding constants were determined at high salt by rapidly mixing various lengths of ssDNA with DCC-PfSSB on stopped-flow apparatus. These included dT35, dT55, dT70 and polydT. The experiments were done at pseudo-first order conditions where the ssDNA was at high excess over PfSSB. The fluorescence time courses were biphasic with a small slow phase. They were therefore fit to two exponentials. For dT70 the second-order binding rate constant was 309 ⁇ " ⁇ "1 , possibly diffusion controlled.
  • the second, slower phase was also observed but this had amplitude less than 20% of the first phase and the observed rate constant for the phase did not follow concentration dependence, suggesting it possibly represents a slow rearrangement.
  • the dissociation rate constant determined from the intercept of y-axis was 7.6 s "1 .
  • the binding rate constant for different lengths of ssDNA are summarised in Table 3.
  • DCC-PfSSB dissociation kinetics from ssDNA were measured done by rapidly mixing the DCC-PfSSB ssDNA complex with a large excess of the unlabelled wtSSB to trap reased ssDNA.
  • Unlabeled SSB has a tighter affinity to ssDNA than its labelled counterpart as was shown by titration assays ( Figure 1C and D). If the PfSSB dissociation takes place as a single step, much slower than binding to the unlabelled trap, the dissociation kinetics should not be affected by the concentration of the wtSSB.
  • Plasmid unwinding by PcrA The activity and possible use of DCC-PfSSB was tested in two double-stranded DNA (dsdDNA) unwinding assays, mediated by helicases.
  • dsdDNA double-stranded DNA
  • PcrA helicase unwound circular plasmids which was first nicked at its double stranded origin of replication by an initiator protein, RepD 4 .
  • RepD covalently binds to the plasmid DNA and increases the processivity of PcrA helicase.
  • DNA70 represents a 70-base stretch of ssDNA, such as dT70.
  • the equilibrium constant for step 2 K 2
  • K 2 the equilibrium constant for step 2
  • SSB 2 .DNA70 which represents the 35-base mode
  • K 2 the equilibrium constant for step 2
  • K 2 the equilibrium constant for step 2
  • SSB 2 .DNA70 the 35-base mode
  • W166C mutation may be made in the Plasm odium ssDNA binding protein.
  • the W166C mutation is made in conjunction with a C93 mutation such as C93 A.
  • approximate 12-15 times signal may be achieved compared to the unbound state.

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Abstract

La présente invention concerne une protéine de la famille des protéines de liaison à l'ADN simple brin (SSB), ladite protéine étant une protéine de Plasmodium modifiée, ladite protéine comprenant au moins une étiquette détectable fixée à un acide aminé de ladite protéine, ledit acide aminé étant situé sur la boucle (L1-1') de résidus assemblant les deux feuillets bêta (β1) et (β1') et les caractéristiques de l'étiquette détectable modifiant la liaison de l'ADN simple brin. L'invention concerne également des acides nucléiques, des utilisations et des procédés correspondants.
PCT/GB2016/052124 2015-07-17 2016-07-14 Détecteur d'adn simple brin Ceased WO2017013400A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018154083A1 (fr) * 2017-02-24 2018-08-30 Universitetet I Tromsø - Norges Arktiske Universitet Protéine de liaison monocaténaire

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5747247A (en) * 1994-07-25 1998-05-05 The Regents Of The University Of California Spectroscopic helicase assay
WO2008152379A1 (fr) * 2007-06-12 2008-12-18 Medical Research Council Protéine marquée de liaison à l'adn simple brin et son utilisation dans un biodétecteur pour la détection et la visualisation d'un adn simple brin.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5747247A (en) * 1994-07-25 1998-05-05 The Regents Of The University Of California Spectroscopic helicase assay
WO2008152379A1 (fr) * 2007-06-12 2008-12-18 Medical Research Council Protéine marquée de liaison à l'adn simple brin et son utilisation dans un biodétecteur pour la détection et la visualisation d'un adn simple brin.

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANTONY EDWIN ET AL: "Plasmodium falciparum SSB Tetramer Binds Single-Stranded DNA Only in a Fully Wrapped Mode", JOURNAL OF MOLECULAR BIOLOGY, vol. 420, no. 4-5, July 2012 (2012-07-01), pages 284 - 295, XP002764378, ISSN: 0022-2836 *
EDWIN ANTONY ET AL: "Plasmodium falciparum SSB Tetramer Wraps Single-Stranded DNA with Similar Topology but Opposite Polarity to E. coli SSB", JOURNAL OF MOLECULAR BIOLOGY, vol. 420, no. 4-5, 1 July 2012 (2012-07-01), United Kingdom, pages 269 - 283, XP055320510, ISSN: 0022-2836, DOI: 10.1016/j.jmb.2012.04.021 *
KOZLOV ALEXANDER G ET AL: "Intrinsically Disordered C-Terminal Tails ofE.coliSingle-Stranded DNA Binding Protein Regulate Cooperative Binding to Single-Stranded DNA", JOURNAL OF MOLECULAR BIOLOGY, vol. 427, no. 4, 3 January 2015 (2015-01-03), pages 763 - 774, XP029136785, ISSN: 0022-2836, DOI: 10.1016/J.JMB.2014.12.020 *
KUNZELMANN SIMONE ET AL.: "Fluorescent biosensors: design and application to motor proteins", EXS, BIRKHAEUSER VERLAG, BASEL, CH, vol. 105, 1 January 2014 (2014-01-01), pages 25 - 47, XP009190657, ISSN: 1023-294X, DOI: 10.1007/978-3-0348-0856-9_2 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018154083A1 (fr) * 2017-02-24 2018-08-30 Universitetet I Tromsø - Norges Arktiske Universitet Protéine de liaison monocaténaire
CN110291208A (zh) * 2017-02-24 2019-09-27 特罗姆瑟大学-挪威北极圈大学 单链结合蛋白
US11447812B2 (en) 2017-02-24 2022-09-20 Universitetet I Tromsø—Norges Arktiske Universitet Single-strand binding protein

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