WO2026005687A1 - Procédé pour établir le profil des modifications covalentes de l'adn au niveau cellulaire ou nucléaire et kit pour utilisation dans ce procédé - Google Patents

Procédé pour établir le profil des modifications covalentes de l'adn au niveau cellulaire ou nucléaire et kit pour utilisation dans ce procédé

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WO2026005687A1
WO2026005687A1 PCT/SE2025/050591 SE2025050591W WO2026005687A1 WO 2026005687 A1 WO2026005687 A1 WO 2026005687A1 SE 2025050591 W SE2025050591 W SE 2025050591W WO 2026005687 A1 WO2026005687 A1 WO 2026005687A1
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dna
fusion protein
modifica
nuclease
ons
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Marek BARTOŠOVIC
Letian ZHANG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • C12Y301/21001—Deoxyribonuclease I (3.1.21.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the present inven ⁇ on refers to thefields of epigene ⁇ cs, genomics, DNA methyla ⁇ on profiling and single-cell and spa ⁇ al omics. More specifically, the present inven ⁇ on refers to a method of profiling covalent DNA modifica ⁇ on at a cellular or nuclei level and a kit for use in the method.
  • Background DNA methyla ⁇ on also known as 5-methylcytosine (5mC) represents a cri ⁇ cal form of covalent DNA modifica ⁇ on.
  • DNA methyla ⁇ on regulates gene ac ⁇ vity and expression within a DNA segment without 10 altering the sequence itself.
  • WGBS Whole-genome bisulfite sequencing
  • WGBS W hen profiling genome-wide DNA methyla ⁇ on, WGBS has to generate a vast amount of data to obtain high quality DNA methyla ⁇ on profiles, leading to high cost of sequencing for each sample (>15,000 kr) and problems with data storage and handling.
  • WGBS does not scale well for large-scale single cell projects (profiling of hundreds of thousands to millions of single-cells and spa ⁇ al resolu ⁇ on).
  • WGBS is not compa ⁇ ble with simultaneous profiling of histone modifica ⁇ ons, an e pigene ⁇ c modifica ⁇ on that has also been explored by the present inventors (Bartosovic et al., 2021, Bartosovic & Castelo-Branco, 2023).
  • WO14190214 discloses a nucleic acid sequence library, wherein a protein-transposome complex binding methylated DNA is disclosed.
  • CN117402938A discloses a whole genome sequencing method, using a transposon compound to fragment DNA.
  • MBD protein is used as a binding protein to target DNA modifica ⁇ on ( 5mC), and that a transposase can be included in a fusion protein.
  • WO2022/056309 discloses in situ genome-wide profiling.
  • an affinity reagent coupled with transposome binds a nucleosome depleted region marker, and can cleave and tag chroma ⁇ n DNA.
  • a fusion protein of an a ffinity reagent and transposase is disclosed, as well as that the nucleo ⁇ de sequence of the tagged DNA segments can be determined.
  • US201213244 discloses methods and composi ⁇ ons for selec ⁇ ve cleavage of nucleic acids with recombinant nucleases.
  • WO2020/167712 discloses chroma ⁇ n mapping 40 assays and kits using long-read sequencing.
  • WO2018/018008 discloses single cell whole genome libraries as well as a nucleosome-depleted nuclei. 1
  • the present inven ⁇ on refers to a method of profiling covalent DNA modifica ⁇ on at a single-cellular or single-nuclei level comprising the steps of: ( a) providing a sample solu ⁇ on, comprising cell(s) or isolated nuclei comprising a nucleus 10 comprising genomic DNA, said genomic DNA comprising or is expected to comprise covalent DNA modifica ⁇ on(s); ( b) adding a buffer comprising a nucleosome deple ⁇ on agent to expose the genomic DNA; (c) adding a fusion protein comprising a DNA modifica ⁇ on binding domain, having the ability to tether the fusion protein to the DNA modifica ⁇ on and a nuclease part; (d) incuba ⁇ ng the fusion protein together with the exposed genomic DNA under condi ⁇ ons allowing the fusion protein to bind to the genomic DNA; ( e) washing the solu ⁇ on of step (d) with buffer to remove any excess fusion protein, i.e.
  • fusion protein that is unbound/untethered; ( f) incuba ⁇ ng the solu ⁇ on of step (e) under condi ⁇ ons allowing the fusion protein to cut the 20 genomic DNA in proximity of the DNA modifica ⁇ on(s); (g) determining the sequence of the fusion-protein incubated DNA of step (f), by high- throughput sequencing thereby iden ⁇ fying the posi ⁇ on(s) of the DNA modifica ⁇ on(s) of the genomic DNA that have been sequenced.
  • the inventors present an alterna ⁇ ve approach to detect genome-wide DNA methyla ⁇ on and/or other DNA modifica ⁇ on with high sensi ⁇ vity.
  • the method is based on recogni ⁇ on of 5mC or any other DNA modifica ⁇ on by a fusion protein comprising a DNA modifica ⁇ on binding d omain, and a nuclease part cu ⁇ ng the DNA. Further, the combina ⁇ on of using a nucleosome deple ⁇ on agent and a fusion protein comprising a DNA modifica ⁇ on binding domain is a unique feature of the present disclosure.
  • a method that is compa ⁇ ble with established droplet-based single-cell pla ⁇ orms (e.g.10X Genomics), facilita ⁇ ng the mapping of DNA methyla ⁇ on at the single-cell or single-nuclei level and holding the poten ⁇ al to achieve spa ⁇ al resolu ⁇ on, is provided. Further, the method can achieve whole-genome coverage but is cost-effec ⁇ ve, compared to the bulk methods such as Infinium Methyla ⁇ on assay and WGBS.
  • nucleosome deple ⁇ on agent genomic DNA is made more accessible (crucial for enhancing recogni ⁇ on by the DNA modifica ⁇ on binding agent, such as methyla ⁇ on binding domain (MBD)), while keeping nuclei intact (essen ⁇ al for the methods scalability).
  • the purpose of nucleosome deple ⁇ on is to remove nucleosomes, but keep the nuclei intact, so they can be used for single-cell profiling or single-nuclei profiling.
  • a fusion protein comprising a DNA modifica ⁇ on binding domain
  • recogni ⁇ on of the DNA modifica ⁇ on,40 such as m5c methyla ⁇ on is facilitated.
  • T hus a key feature of the inven ⁇ on is the incorpora ⁇ on of nucleosome deple ⁇ on prior to transposi ⁇ on. Without this step, the MBD-Tn5 has limited access to methylated regions, and is 2
  • the fusion protein has the ability to tagment the modified bases of the genomic DNA to be profiled.
  • the nuclease part is a transposase part comprising sequencing linkers and a means for detec ⁇ on that upon binding have the ability to be inserted to any genomic regions enriched in modified bases of the genomic DNA to be profiled, to insert the sequencing linkers and 10 the means for detec ⁇ on to the modified bases of the genomic DNA to be profiled, and to be used for determining the sequence of the fusion protein incubated DNA.
  • the nuclease part is a transposase (Tn) part which inserts sequencing linkers and a detectable barcode into the genomic DNA in proximity to the modified bases.
  • the inserted barcode uniquely iden ⁇ fies the DNA modifica ⁇ on or modality.
  • the covalent DNA modifica ⁇ on to be profiled is chosen from methyla ⁇ on, p referably 5-methylcytosine (5mC).
  • p referably 5-methylcytosine (5mC).
  • the method of the present inven ⁇ on would be used for whole genome analysis. However, profiling of parts or specific regions of the genome is also possible.
  • the nucleosome deple ⁇ on agent is chosen from lithium assisted nucleosome 20 deple ⁇ on (LAND) or alterna ⁇ ve detergents, such as SDS.
  • the nucleosome deple ⁇ on agent is LAND.
  • the DNA modifica ⁇ on binding domain of the fusion protein is a methyla ⁇ on binding domain (MBD), chosen from MBD1, MBD2, MBD3, MBD4, MBD5, MBD6, MBD7, MBD8, MBD9, MBD10, MBD11, MeCP2, BAZ2A, BAZ2B, SETB1 and SETB2.
  • MBD1 or MBD2 MBD1 or MBD2.
  • the assay is simplified, obvia ⁇ ng the need for any intermediate an ⁇ body binding steps.
  • the nuclease part of the fusion protein is a Micrococcal nuclease (MNase) or DNase I.
  • the nuclease part is a transposase part (Tn), chosen from any transposase (Tn), preferably Tn5 or Tn7.
  • Tn transposase part
  • the means for detec ⁇ on of the nuclease part comprises a unique barcode.
  • these are 8-nucleo ⁇ de sequences, designed to iden ⁇ fy modali ⁇ es (in mul ⁇ modal profiling t ogether with histone modifica ⁇ ons or profiling of other modifica ⁇ ons).
  • the barcode is a transposase-specific DNA barcode.
  • the method of the present inven ⁇ on is for prepara ⁇ on of a sequencing library comprising profiling informa ⁇ on of covalent DNA modifica ⁇ ons, such as DNA methyla ⁇ on.
  • the method is for mul ⁇ modal profiling.
  • the mul ⁇ modal profiling comprises profiling of DNA methyla ⁇ on together with open chroma ⁇ n (ATAC-seq) or histone modifica ⁇ ons.
  • the method can simultaneously detect DNA methyla ⁇ on and open chroma ⁇ n (ATAC-seq) through transposase-specific DNA barcode.
  • the method can simultaneously detect DNA methyla ⁇ on and histone 10 modifica ⁇ ons (CUT&Tag) through transposase-specific DNA barcodes.
  • the method of the present inven ⁇ on is suitable for use in applica ⁇ ons related to single-cell resolu ⁇ on, such as on 10X Genomics, or single-cell indexing methods, such as combinatorial indexing.
  • the method of the present inven ⁇ on is suitable for use in applica ⁇ ons related to spa ⁇ al resolu ⁇ on, such as DBiT-sequencing or Visium, wherein mul ⁇ ple cells or regions of interest within a ⁇ ssue are profiled in parallel.
  • the present inven ⁇ on relates to a kit for use in DNA methyla ⁇ on profiling at single-cell or single-nuclei level of genomic DNA, comprising (i) reagents for use in lithium assisted n ucleosome deple ⁇ on and (ii) an MBD-nuclease fusion protein, comprising a DNA methyla ⁇ on 20 binding domain and a nuclease part.
  • the MBD-Tn fusion protein is chosen from MBD1-MNase, MBD2-MNase, MBD1-DNase I, MBD2-DNase I, MBD1-Tn5, MBD1-Tn7, MBD2-Tn5 or MBD2-Tn7, for recogni ⁇ on of 5mC methylated DNA.
  • the fusion protein is an MBD1-Tn5 or MBD1-Tn7 fusion protein for recogni ⁇ on of methylated DNA.
  • the nuclease part comprises a transposase part, wherein the transposase part comprises sequencing linkers and means for detec ⁇ on, wherein the means for detec ⁇ on comprises unique barcodes.
  • the kit comprises: 30 (i) a DEFND buffer for use in lithium assisted nucleosome deple ⁇ on, comprising NIB buffer and lithium diiodosalicylate; ( ii) a binding buffer, for use in incuba ⁇ on of MBD-nuclease fusion protein together with a nucleosome-depleted nuclei; ( iii) an MBD-nuclease fusion protein; (iv) op ⁇ onally a wash buffer; (v) a tagmenta ⁇ on buffer; (vi) op ⁇ onally reagents for construc ⁇ on of sequencing library; and (vii) instruc ⁇ ons for use. 4
  • the kit is suitable for use in mul ⁇ modal profiling, comprising profiling of DNA methyla ⁇ on together with open chroma ⁇ n (ATAC-seq) or histone modifica ⁇ on.
  • the present inven ⁇ on is a novel method to profile DNA methyla ⁇ on using a combina ⁇ on of Lithium Assisted Nucleosome Deple ⁇ on (LAND) and methyla ⁇ on binding d omain (MBD)-Tn5 transposase fusion protein. This method is poten ⁇ ally commercialized as a kit or service.
  • nucleosome deple ⁇ on such as Lithium Assisted Nucleosome D eple ⁇ on (LAND)
  • LAND Lithium Assisted Nucleosome D eple ⁇ on
  • MBD methyla ⁇ on binding domain
  • MBD-Tn5 Tn5 transposase
  • MBD domain facilitates recogni ⁇ on of methylated DNA (5mC), whereas Tn5 transposase inserts sequencing linkers and a barcode into genomic DNA through cut and paste mechanisms.
  • Nucleosome deple ⁇ on treatment such as LAND treatment, is crucial for enhancing the recogni ⁇ on of 5mC by MBD-Tn5 fusion proteins within the genomic DNA. Maintaining intact nuclei is also essen ⁇ al for the method's scalability.
  • the method is compa ⁇ ble with established droplet-based single-cell pla ⁇ orms (e.g.10x Genomics), facilita ⁇ ng the mapping of DNA methyla ⁇ on at the single-cell level and holding the poten ⁇ al to achieve spa ⁇ al resolu ⁇ on.
  • This method of the inven ⁇ on can yield data of comparable quality to that of WGBS but the 20 sequencing depth requirement and cost is much less.
  • T he principles of the method of the inven ⁇ on can be used to develop kit(s) for DNA methyla ⁇ on sequencing library preps in bulk, single-cell, and/or spa ⁇ al analysis. The method can also be used as a service to support the kits.
  • the present inven ⁇ on is unique in that it introduces the combina ⁇ on of (1) a methyla ⁇ on- binding fusion transposase, and (2) a chroma ⁇ n prepara ⁇ on strategy to remove nucleosomes.
  • This combina ⁇ on provides a synergis ⁇ c effect which is both non-obvious and func ⁇ onally advantageous. Effects and features of the second aspect are to a large extent analogous to those described above in 30 connec ⁇ on with thefirst aspect. Embodiments men ⁇ oned in rela ⁇ on to thefirst aspect are largely compa ⁇ ble with the second aspect. The present disclosure will become apparent from the detailed descrip ⁇ on given below.
  • F igure 1 Genome browser view shows DNA methyla ⁇ on profiles obtained by MBD-Tn5 without LAND, MBD-Tn5 with LAND, and WGBS.
  • Figure 2 Sequencing depth required for MBD-Tn5 with LAND and WGBS methods to obtain the data shown in Figure 1.
  • F igure 3 Schema ⁇ c view of the method of the present inven ⁇ on. (a) LAND and (b) MBD-Tn5 10 incuba ⁇ on.
  • Figure 4 Example of sequencing library structure.
  • F igure 5 Library structure ((a)-(h)). Defini ⁇ ons
  • the term “single-cell level” refers to the level of an individual cell, i.e. analysis of the single-cell level refers to evalua ⁇ on of the individual cell. “Single-cell resolu ⁇ on” thus provides informa ⁇ on about the individual cells, and “single-cell sequencing” examines the nucleic acid sequence informa ⁇ on from individual cells I n analogy, the term “single-nuclei level” refers to the level of an individual cell nucleus.
  • the term “spa ⁇ al” refers to a specific area or a region within a ⁇ ssue, and thus to informa ⁇ on (such 20 as gene expression informa ⁇ on) obtained from such areas or regions, e.g. informa ⁇ on about cell-cell interac ⁇ ons.
  • CpG refers to “5’-C-phosphate-G-3’”, that is, cytosine and guanine separated by only one phosphate group.
  • CpG islands (“CGIs”) refer to regions of the genome that contain a large number of CpG dinucleo ⁇ de repeats. CGIs are usually associated with lack of DNA methyla ⁇ on, and thus “CpG density” is inversely correlated with DNA methyla ⁇ on.
  • profiling in the context of this disclosure, refers to the process of obtaining a specific DNA pa ⁇ ern (a “profile”) from a sample of cells or ⁇ ssue of a person, pa ⁇ ent and/or ⁇ ssue.
  • tagmenta ⁇ on refers to an ini ⁇ al step in DNA library prepara ⁇ on where unfragmented DNA is cleaved and tagged for analysis. Typically, such cleavage and tagging can be performed by30 using an enzyme or fusion protein having transposase ac ⁇ vity.
  • a “nuclease” includes any enzyme having the capacity to cleave/cut a DNA molecule
  • transposase refers to a nuclease also having the capacity to insert a sequence and/or to be inserted into the DNA molecule cleaved.
  • a transposase has both a nuclease ac ⁇ vity and an inser ⁇ on/tagging ac ⁇ vity.
  • high-throughput sequencing refers to technologies that sequence DNA (and RNA) in a rapid and cost-effec ⁇ ve manner, also known as next-genera ⁇ on sequencing. Any sequencing methods exemplified in this disclosure is included in this concept. 6
  • NIB buffer refers to a nuclei isola ⁇ on buffer.
  • DEFND buffer refers to a specific LAND buffer.
  • the present inven ⁇ on thus provides methods and kits for improved DNA modifica ⁇ on profiling at a single-cellular, single-nuclei or spa ⁇ al level, wherein the combina ⁇ on of (i) using a nucleosome deple ⁇ on reagent, thereby making the genomic DNA more accessible, while keeping the nuclei intact, 10 with (ii) incuba ⁇ ng the genomic DNA with a fusion protein comprising a modifica ⁇ on binding domain and a nuclease part, allowing binding and subsequent inser ⁇ on of suitable sequencing linkers and m eans for detec ⁇ on, results in advantages such as higher sensi ⁇ vity and improved scalability compared to prior art methods.
  • a sample solu ⁇ on comprising cell(s) and/or nuclei to be analysed which cells comprises a nucleus comprising genomic DNA.
  • the cells are typically of eukaryo ⁇ c origin, such as plant or animal cells, such as human cells or mouse cells. Any types of ⁇ ssues may be used, such as brain heart, muscle, lung and blood.
  • the cells are provided in a solu ⁇ on and/or in any form that can be solubilized.
  • the genomic DNA of the cells contain or are expected to contain covalent DNA modifica ⁇ ons in at least a part of the genomic DNA.
  • the profiling process is made for the whole genome, and in s ome embodiments, it is concentrated to a part or region of the genomic DNA that are of special interest.
  • the DNA modifica ⁇ ons can be chosen from various type of modifica ⁇ ons, such as methyla ⁇ on, i ncluding 5-methylcytosine modifica ⁇ on.
  • other modified bases exist in DNA, for example 5- hmC, 5-fC,6-mA. Any DNA modifica ⁇ on might be detected by the principle of the present inven ⁇ on, e .g. LAND + fusion of 5-hmC/5-fC/6-mA reader to Tn5.
  • hmC readers are for example Uhrf2, Thy28 and Wdr76 (see also h .
  • 6mA readers are YTHDF1, YTHDF2 and YTHDC5.
  • a buffer comprising a nucleosome deple ⁇ on agent thereby forming a cell suspension.
  • the nucleosome deple ⁇ on agent has the capacity to remove the nucleosomes, make the genomic DNA more accessible, while keeping the nuclei intact.
  • the nucleosome deple ⁇ on agent can be chosen from any agent having the desired effect, such as s odium dodecyl sulfate (SDS).
  • the nucleosome deple ⁇ on agent is chosen from lithium assisted nucleosome deple ⁇ on (LAND) or alterna ⁇ ve detergents, such as SDS, p referably LAND.
  • LAND lithium assisted nucleosome deple ⁇ on agent
  • a concentra ⁇ on of lithium diiodosalicylate in the 40 interval of 1 mM to 50 mM can be used, such as about 12,5 mM.
  • incuba ⁇ on ⁇ me e.g. when using a working concentra ⁇ on of 12,5 mM, can be in the interval of 30 seconds to 10 minutes, such as about 5 minutes.
  • alterna ⁇ ve detergents for example cross-linking and SDS treatment, b ut other alterna ⁇ ves might exist as well.
  • the nuclei need to befixed using formaldehyde.
  • a specifically designed fusion protein is added to and incubated with the suspension, so that the modifica ⁇ on binding domain of the fusion protein is allowed to bind to the e xposed genomic DNA, typically the nuclei.
  • This incuba ⁇ on is typically performed overnight, and can also be done for about 10 minutes or for a few hours,
  • the temperature is typically about 4 ⁇ C, and can10 also be up to about room temperature, as long as proper binding is achieved.
  • the incuba ⁇ on can be performed as exemplified in the Example sec ⁇ on. Typically, incuba ⁇ on is performed in a horizontal roller at 4 degrees overnight. However, other temperatures and incuba ⁇ on c ondi ⁇ ons could also work, as long as the fusion protein is allowed to bind to the DNA modifica ⁇ on sites that are available and to be profiled. For example, the incuba ⁇ on can be performed at room temperature at a ⁇ me interval ranging from 10 minutes to 2 hours.
  • the fusion protein comprises at least two parts: one part comprising a DNA modifica ⁇ on binding func ⁇ onality, so that the fusion protein binds to the DNA modifica ⁇ ons to be profiled.
  • DNA 20 modifica ⁇ ons could be of any type, such as 5-methylcytosine methyla ⁇ on (5mC), for which type of modifica ⁇ on the present inven ⁇ on is especially suited.
  • the DNA modifica ⁇ on binding p art is a methyla ⁇ on binding domain (MBD), such as a methyla ⁇ on binding domain chosen from MBD1, MBD2, MBD3, MBD4, MBD5, MBD6, MBD7, MBD8, MBD9, MBD10, MBD11, MeCP2, BAZ2A, B AZ2B, SETB1 and SETB2, preferably MBD1 or MBD2.
  • MBD domains There are currently 11 MBD domain proteins that have a bit different sequence of MBD domains. It is possible to use any MBD domains to engineer MBD-Tn fusion proteins, such as MBD1-Tn5 and generate good quality data.
  • the fusion protein also comprises a nuclease part, t hat comprises a nuclease, that has the ability to cut the genomic DNA.
  • the nuclease part typically 30 comprises sequencing linkers and means for detec ⁇ on, whereby the transposase part has the ability to break the genomic DNA and insert the sequencing linkers and means for detec ⁇ on into the genomic DNA, at or close to the posi ⁇ on of a DNA modifica ⁇ on, thereby facilita ⁇ ng recogni ⁇ on of the DNA modified posi ⁇ on.
  • the nuclease part can be any nuclease having the desired effect.
  • a transposase (Tn) has been shown to be especially advantageous for the purposes of the present i nven ⁇ on, especially when profiling methyla ⁇ ons.
  • the nuclease is chosen from any transposase (Tn) or nuclease having similar func ⁇ on, such as Micrococcal nuclease (MNase) or DNase I, or a transposase chosen from Tn5 or Tn7.
  • MBD-Tn fusion proteins it is also an op ⁇ on to fuse the MBD domain and other Transposases such as Tn5 or Tn7 or other nucleases such as MNase or DNase I to have similar results.
  • Fusing MBD-Tn5 with more domains such as a DNA- 40 binding domain of transcrip ⁇ on factors (DBD) is also expected to be able to detect transcrip ⁇ on f actor binding sites, without the effects of chroma ⁇ n context.
  • DBD transcrip ⁇ on factors
  • the fusion protein can comprise a nuclease having the capacity to cleave (cut) the DNA molecule at certain posi ⁇ ons, or it can comprise a nuclease having transposase ac ⁇ vity, i.e.
  • transposase that has the capacity to cut and paste the DNA molecule at certain posi ⁇ ons, i.e. it basically does two things at the same ⁇ me.
  • the c ut pieces of DNA can be purified, but sequencing linkers have to be purified and linkers ligated by 10 e.g. DNA ligase.
  • a ⁇ er having incubated the fusion protein together with the exposed genomic DNA, in order to have the modifica ⁇ on binding domain of the fusion protein to bind to, or close to, modifica ⁇ ons occurring in the genomic DNA (i.e.
  • the solu ⁇ on of fusion protein together with exposed g enomic DNA is washed to remove any excess fusion protein.
  • the washing procedure is performed at least twice.
  • the suspension including fusion protein is washed to remove excess fusion protein that is unbound/untethered to the DNA.
  • a tagmenta ⁇ on step is then performed, wherein the fusion protein is incubated with the cell 20 suspension that has undergone nucleosome deple ⁇ on, so that the fusion protein can cut the DNA and insert the sequencing linkers and means for detec ⁇ on at the posi ⁇ ons of the modifica ⁇ on to be profiled.
  • the washed solu ⁇ on is incubated under condi ⁇ ons ac ⁇ va ⁇ ng the nuclease part of the fusion protein, thereby allowing the fusion protein to cut the genomic DNA in proximity of the DNA modifica ⁇ on(s).
  • This incuba ⁇ on step is also referred to as tagmenta ⁇ on in some embodiments o f the present disclosure.
  • the tagmenta ⁇ on step is performed at a higher temperature than in the previous incuba ⁇ on, typically at about 37 ⁇ C.
  • varia ⁇ ons may occur, as long as condi ⁇ ons ac ⁇ va ⁇ ng the nuclease/transposase func ⁇ on of cu ⁇ ng or cu ⁇ ng and pas ⁇ ng the DNA is obtained.
  • the fusion protein cuts the genomic DNA in proximity of the DNA modifica ⁇ on(s) to which 30 it has bound, which typically is within about 50-100 bp from the modifica ⁇ on(s), so that for example about 150 bp fragments with high modifica ⁇ on enrichment are obtained.
  • T he sequencing linkers are used for PCR amplifica ⁇ on of the library. Examples of the PCR amplifica ⁇ on procedure and sequencing linkers adapted for use with Tn5 is provided in the Example sec ⁇ on. Typically, one part of the sequencing linkers is a so-called mosaic-end adapter, which is required for recogni ⁇ on by the transposase, such as Tn5.
  • the second part of the sequencing linker serves for sequencing primers annealing.
  • the means for detec ⁇ on which in this context 40 comprises iden ⁇ fica ⁇ on of modality (DNA methyla ⁇ on, open chroma ⁇ n or histone modifica ⁇ on), such as barcodes, can be posi ⁇ oned in between these sequencing linker parts.
  • the means for detec ⁇ on can be any molecule or part that provide the desired effect of allowing detec ⁇ on and/or iden ⁇ fica ⁇ on of the profiled DNA modifica ⁇ on(s).
  • the means for detec ⁇ on is integrated in the nuclease part in the form of a unique barcode.
  • the unique barcode may for example be posi ⁇ oned in between the parts of the sequencing linker.
  • the unique barcodes are cons ⁇ tuted of 8-nucleo ⁇ de sequences, designed to iden ⁇ fy modali ⁇ es (in mul ⁇ modal profiling t ogether with histone modifica ⁇ ons). However, varia ⁇ ons are included.
  • any length that allows to dis ⁇ nguish modali ⁇ es typically 6-8 nucleo ⁇ des, and in some embodiments as short as 2 nucleo ⁇ des or as long as 15 or 20 nucleo ⁇ des, or even longer, are also included.
  • the sequence of the genomic DNA at posi ⁇ ons where the fusion protein has inserted s equencing linkers is determined, thereby iden ⁇ fying the posi ⁇ ons of the genomic DNA including modifica ⁇ ons of the type that is intended to be iden ⁇ fied. This can be achieved as exemplified in the Example sec ⁇ on.
  • Illumina short read sequencing which is primarily used by the present inventors
  • MGI short read nanopore long read
  • pac bio long read sequencing and similar sequencing methods, including high-throughput sequencing methods and/or next genera ⁇ on sequencing (NGS) methods.
  • NGS next genera ⁇ on sequencing
  • Applica ⁇ ons The method and kit of the inven ⁇ on can be used in various applica ⁇ ons related to profiling of DNA modifica ⁇ ons, such as methyla ⁇ on, at a single-cellular, single-nuclei and/or spa ⁇ al level.
  • the method of the inven ⁇ on is used for prepara ⁇ on of a sequencing library comprising profiling informa ⁇ on of covalent DNA modifica ⁇ ons, such as DNA methyla ⁇ ons.
  • the method and kit of the inven ⁇ on is used for applica ⁇ ons related to single- cell resolu ⁇ on, such as on 10X Genomics, or single-cell indexing methods, such as combinatorial indexing. However, other applica ⁇ on related to single-cell resolu ⁇ on are also included.
  • the method and kit of the inven ⁇ on is used for applica ⁇ ons related to spa ⁇ al resolu ⁇ on.
  • Spa ⁇ al resolu ⁇ on may for example be accomplished using DBiT-seq (h ⁇ ps://www.nature.com/ar ⁇ cles/s41586-023-05795-1) or other spa ⁇ al methods, such as Visium, w herein mul ⁇ ple cells are profiled in parallel. However, other applica ⁇ ons related to spa ⁇ al resolu ⁇ on are also included. 30
  • the method is used for mul ⁇ modal profiling.
  • the mul ⁇ modal profiling comprises profiling of DNA methyla ⁇ on together with open chroma ⁇ n (ATAC- seq) or histone modifica ⁇ ons. More than two modes are possible to profile using the method of the i nven ⁇ on.
  • modifica ⁇ on alterna ⁇ ves than those listed here are also included.
  • at least two 2 histone modifica ⁇ ons could be profiled simultaneously, and in some embodiments up to 10, 20, 30, 40 or 50 simultaneous modifica ⁇ ons.
  • the upper theore ⁇ cal limit would be defined by c rowding of the Tn5/Tn7 inser ⁇ ons in the DNA in case of overlapping features/modali ⁇ es.
  • Applica ⁇ ons related to single-nuclei resolu ⁇ on are also included using the method and kit of the present inven ⁇ on.
  • the present inven ⁇ on refers to a kit for use in DNA methyla ⁇ on profiling at single- cell or single-nuclei level of genomic DNA.
  • the kit would typically comprise the components/ingredients that are necessary for performing the method for any sample, at least including (i) reagents for use in lithium assisted nucleosome deple ⁇ on and (ii) a MBD-nuclease fusion protein, comprising a DNA methyla ⁇ on binding domain and a nuclease part.
  • the fusion protein is chosen from fusion proteins that are based on MBD1 or MBD2 together with a Tn part chosen from Tn5 or Tn7, or a nuclease part chosen from MNase or DNase I, thereby allowing recogni ⁇ on of m5c methylated DNA.
  • the nuclease part comprises a transposase part, wherein the 10 transposase part comprises sequencing linkers and means for detec ⁇ on, wherein the means for detec ⁇ on comprises unique barcodes, as disclosed and discussed in other parts of this applica ⁇ on. Such sequencing linkers and barcodes are included here.
  • the kit would include addi ⁇ onal components and/or ingredients, such as: - a DEFND buffer for use in lithium assisted nucleosome deple ⁇ on, comprising NIB buffer and lithium diiodosalicylate; - a binding buffer (also called an ⁇ body buffer in the context of this disclosure), for use in incuba ⁇ on of MBD-nuclease fusion protein together with a nucleosome-depleted nuclei; - an MBD-nuclease fusion protein; - a wash buffer, for washing away non-binding/excess fusion protein; 20 - a tagmenta ⁇ on buffer; - op ⁇ onally reagents for sequencing; and - instruc ⁇ ons for use.
  • a DEFND buffer for use in lithium assisted nucleosome deple ⁇ on, comprising NIB buffer and lithium diiodosalicylate
  • - a binding buffer also called an ⁇ body buffer in the context of this disclosure
  • the kit would be adapted for mul ⁇ modal profiling. comprising profiling of DNA methyla ⁇ on together with open chroma ⁇ n (ATAC-seq) or histone modifica ⁇ ons.
  • Some components/ingredients would be generic, i.e. to be used for any sample, regardless of cell type, modifica ⁇ on type and genomic DNA sequences. This would typically include buffers to be used a s well as MBD-nuclease fusion protein including sequencing linkers and unique barcodes (means for 30 detec ⁇ on).
  • reagents for sequencing would be generic, as long as informa ⁇ on on the specific sample DNA sequences is not required.
  • Some components/ingredients could however be specific, i.e. dependent on the specific sample and/or its genomic DNA sequence to be profiled, and may t herefore not be suitable to be included in a generic kit, but could be included in a kit adapted for profiling of a specific type of sample, cell, modifica ⁇ on type and/or genomic DNA.
  • the present disclosure will now be described with reference to the following examples.
  • EXAMPLES E xample 1 - Lithium-assisted nucleosome deple ⁇ on (LAND) followed by Tn5-based DNA methyla ⁇ on profiling 11
  • Buffer prepara ⁇ on N IB buffer (for LAND), prepare fresh 1 00 mM lithium diiodosalicylate (396 g/mol): Weigh 7.92 mg of lithium diiodosalicylate and dissolve in 200 ⁇ l Nuclease-free Water. Weigh chemicals first and add water later in a proportion. Stock solution can be stored at -20 C.
  • DEFND buffer D EFND buffer comprises 175 ⁇ l NIB and 25 ⁇ l 100 mM lithium diiodosalicylate.
  • 2X Wash buffer 10 Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 13 ⁇ L 2 M spermidine, add 1 Roche Complete Protease I nhibitor EDTA-Free tablet, bring the final volume to 25 mL with nuclease free water. Store the buffer at 4 °C for up to 1 week.
  • Digitonin300-Wash Mix 1 mL 2X Wash Buffer, 60 ⁇ L 5 M NaCl, 20 ⁇ L Digitonin 5%, 2 ⁇ L of 10% NP-40, 200 ⁇ L 20% BSA, bring thefinal volume to 2 mL with nuclease free water.
  • Make fresh. Prepare 500 ⁇ L per sample. * 150mM in wash + 150mM supplemented here Tagmentation buffer Mix 750 ⁇ L 2X Wash buffer, 15 ⁇ L 1 M MgCl2 (to 10 mM), 45 ⁇ L 5 M NaCl, 15 ⁇ L 5% Digitonin, 1.5 ⁇ L 10 of 10% NP-40, 150 ⁇ L 20% BSA, bring the final volume to 1.5 mL with nuclease free water. Make fresh. Prepare 250 ⁇ L per sample. 13
  • 2X TD buffer Mix 20 ⁇ L 1M Tris pH 7.5, 10 ⁇ L 1 M MgCl2 (to 10 mM), 200 ⁇ L 100% dimethylformamide, bring the final volume to 1 mL with dH2O. Store the buffer at -20 °C for up to 1 year. Detailed protocol 1 .
  • LAND treatment Tip Each LAND-treated sample requires 200,000 cells, If processing more samples, pool samples t ogether, scale up buffers (e.g.3 samples using 600 ⁇ l DEFND buffer but NIB buffer can be 10 ml), 10 and split cells at step f (e.g.3 samples using approximately 330 ⁇ l antibody buffer for resuspension).
  • Fusion protein incubation a) Add 1 ⁇ L MBD fusion proteins loaded with specific barcodes (such as MBD1-Tn5 Barcode A) into the 100 ⁇ l nuclei suspension and mix thoroughly. b) Place the tubes on a horizontal roller and incubate at 4°C overnight. 3. Nuclei washing and Tn5 (P5) tagmentation a) Next day, centrifuge for 3 min at 600g at 4°C and discard the supernatant. Use a swinging 30 bucket rotor centrifuge for all nuclei handling steps. b ) Resuspend the nuclei in 200 ⁇ L of Dig-300 wash buffer. c) Repeat previous steps for a total of 2 washes and remove the supernatant carefully. d) Resuspend the nuclei pellet in 200 ⁇ L of Tagmentation buffer. Pipette mix five times with 200 ⁇ L pipette tip to resuspend gently the pellet. 14
  • Tagmented gDNA purification using Zymo kit 10 a) Add 500 ⁇ L DNA binding buffer to the tube (Zymo DNA Clean & Concentrator-5) and transfer the mix into zymo kit column (pre-assembled with bottom waste collection tube) b ) Centrifuge for 1min at 12,000 xg. Discard the flow-through. c) Add 200 ⁇ L of zymo wash buffer to the column. Centrifuge for 1 min at 12,000 xg. Discard the supernatant. d ) Repeat the wash step once more. e) Perform dry spin – After discarding the supernatant, centrifuge the assembled columns once more for 1 minute at 12,000 xg f ) Transfer the column into a clean 1.5ml eppendorf tube.
  • 10X_LA_primer_noBCD (10 ⁇ M) a ) Run the following program in a PCR cycler * Number of PCR cycles can be determined by using 1 uL of P7-tagmented product in qPCR 8.
  • Library purification using SPRI beads a) Purify the product using 1.2x SPRI beads as described before.
  • c Verify the size distribution of the library by a capillary electrophoresis (e.g Agilent Bioanalyzer High Sensitivity kit following manufacturer's instructions).
  • Nuclei washing and Tn5 (P5) tagmenta ⁇ on a Next day, centrifuge for 3 min at 600g at 4°C and discard the supernatant. Use a swinging bucket rotor centrifuge for all nuclei handling steps.
  • b Resuspend the nuclei in 200 ⁇ L of Dig-300 wash buffer.
  • c) Repeat previous steps for a total of 2 washes and remove the supernatant carefully.
  • d) Resuspend the nuclei pellet in 200 ⁇ L of Tagmenta ⁇ on buffer. Pipe ⁇ e mixfive ⁇ mes with 200 20 ⁇ L pipe ⁇ e ⁇ p to resuspend gently the pellet.
  • e Incubate at 37°C for 1 h at a heater block.
  • Example 3 The inventors have compared the DNA methylation profiles obtained by the combination of MBD-Tn5 without LAND (track 1) and with LAND (track 2) to those of WGBS (track 3) in K562 cells ( Figure 1). MBD-Tn5 + LAND method recapitulates the DNA methylation pattern obtained by WGBS in human 19
  • MBD-Tn5 and LAND method only need 5 – 30 million reads to obtain a similar DNA methylation pattern as WGBS which requires at least 500 million reads (Figure 2).
  • E xample 4 – Fusion protein sequences Below are examples of fusion proteins used in the context of the present invention disclosed.
  • Sequencing linkers and barcodes are not included here (but can be seen as part of the DNA sequences in Example 5): 10 20 30 L inker (SEQ ID NO: 5): GGGGSGGGGSGGGGSGGGGS
  • SEQ ID NO: 5 For the following fusion protein sequences, the corresponding parts (His + 3xFLAG tag (SEQ ID NO: 2), Tn5 transposase (SEQ ID NO:4) and linker (SEQ ID NO: 5)) apply as for psfMeCP2Tn5-c001.
  • the modifica ⁇ on binding domain varies between the fusion proteins.
  • Cluster regenera ⁇ on add customer index2 primer to sequence the second index (i5) (top strand as template, 48 cycles, this is cell barcode)
  • C ustomer primer I2 5'- CTGTCTCTTATACACATCTGCCGTCCTCGATCGC -3' 4.
  • Add Nextera Read 2 primer to sequence the second read top strand as template, 36 cycles, DNA methyla ⁇ on read): N extera primer R2: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3' 20
  • Final library structure of DNA methyla ⁇ on and histone modifica ⁇ ons mul ⁇ -modal in bulk (fig 5 h) 25
  • Sequencing primers in bulk are the same as in the single cells. Sequencing cycles can change to Read1: 50 cycles, Read2: 50 cycles, Index1: 8 cycle, and Index2: 8 cycle. References Yong, WS., Hsu, FM. & Chen, PY. Profiling genome-wide DNA methylation. ⁇ Epigenetics & Chromatin ⁇ 9, 26 (2016). https://doi.org/10.1186/s13072-016-0075-3 O lova, N., Krueger, F., Andrews, S. ⁇ et al. ⁇ Comparison of whole-genome bisulfite sequencing library 10 preparation strategies identifies sources of biases affecting DNA methylation data. ⁇ Genome Biol ⁇ 19, 33 (2018).

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Abstract

La présente invention concerne un procédé pour établir le profil des modifications covalentes de l'ADN au niveau cellulaire ou nucléaire, incluant les étapes suivantes : (a) mise à disposition d'une solution échantillon, comportant une ou plusieurs cellules contenant un noyau contenant de l'ADN génomique; (b) ajout d'un tampon contenant un agent d'appauvrissement des nucléosomes afin d'exposer l'ADN génomique; (c) ajout d'une protéine de fusion comportant un domaine de liaison à la modification de l'ADN et une partie nucléase; (d) incubation de la protéine de fusion avec l'ADN génomique exposé dans des conditions permettant à la protéine de fusion de se lier à l'ADN génomique; (e) lavage de la solution de l'étape (d) avec un tampon pour éliminer tout excès de protéine de fusion; incubation de la solution de l'étape (e) dans des conditions permettant à la protéine de fusion de couper l'ADN génomique à proximité de la ou des modifications de l'ADN; et identification de la séquence de l'ADN incubé avec la protéine de fusion de l'étape (e) par séquençage haut débit, permettant ainsi d'identifier la ou les positions de la ou des modifications de l'ADN génomique qui ont été séquencées. Selon d'autres aspects, l'invention concerne également un kit pour utilisation dans la mise en œuvre du procédé.
PCT/SE2025/050591 2024-06-27 2025-06-19 Procédé pour établir le profil des modifications covalentes de l'adn au niveau cellulaire ou nucléaire et kit pour utilisation dans ce procédé Pending WO2026005687A1 (fr)

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