Capturing methylated nucleic acids

Abstract

The present invention provides methods and compositions for capturing and isolating methylated DNA from a sample using a polypeptide comprising a methyl-CpG-binding domain.

Description

[0001] Sequence Listing

[0002] The "Sequence Listing XML" is submitted herewith in XML file format, and (i) the name of the file is WMG-010-01WO.xml; (ii) the creation date is March 16, 2023; and (iii) the size of the file is 7,481 bytes, and the contents in the XML file are incorporated by reference. Technical Field

[0003] The present disclosure generally relates to methods and compositions for capturing methylated nucleic acids using capture moieties comprising a methyl-CpG-binding domain. Background Art

[0004] Studies have shown that epigenetics can play a key role in disease. One of the more commonly studied epigenetic modifications is DNA methylation. DNA methylation is believed to contribute to regulating gene expression. Therefore, many diseases (such as cancer) have been associated with the increase of DNA methylation levels. In cells, most DNA methylation occurs by adding methyl groups on the 5' carbon (5mC) of cytosine residues, particularly on the cytosine found in the extended segments (CpG islands) of CpG dinucleotides. It is believed that in some cases, increased methylation (hypermethylation) is a natural response to certain diseases (for example, cancer), which helps to reduce the transcription of certain genes. In cells, methylated DNA can reduce transcription and / or can recruit repressor complexes to genes by preventing the binding of certain transcription factors.

[0005] Due to its emerging role in cancer, there is increasing interest in assessing the methylation status of genetic material from cancer cells (e.g., tumor cells). cfDNA from cancer cells (such as circulating tumor DNA (ctDNA)) has a different methylation status compared to other cell-free DNA (cfDNA). Therefore, it represents a promising approach for cancer diagnosis and research. Summary of the invention

[0006] The present invention provides methods and compositions for capturing and separating methylated DNA from a sample using a polypeptide comprising a methyl-CpG-binding domain. In a preferred aspect, the captured DNA is cell-free DNA (cfDNA), such as circulating tumor DNA (ctDNA). ctDNA is an important biomarker for assessing and diagnosing cancer and is easily identified from other cfDNA due to its methylation state. In addition, the methylation profile of ctDNA (including any changes) can be used to detect, classify and monitor the progress of cancer or its treatment.

[0007] Although methods exist for capturing and isolating methylated DNA, these existing methods (such as a process known as methylated DNA immunoprecipitation sequencing (MeDIP-seq), in which methylated immunoprecipitation is coupled to next-generation sequencing) require high concentrations of DNA and / or are unable to capture and isolate short nucleic acids. However, ctDNA is typically short in length (typically less than 200 bp) and is found at low concentrations. The methods and compositions disclosed herein using polypeptides comprising a methyl-CpG-binding domain (MBD) are able to capture short methylated DNA sequences present at low concentrations.

[0008] The naturally occurring methyl-CpG binding domain protein 2 (MBD2) has the ability to specifically bind to 5mC, especially when 5mC is present in CpG islands. However, the present inventors have discovered that a large number of polypeptides comprising a methyl-CpG-binding domain (including polypeptides engineered from the naturally occurring MBD2 sequence) are capable of capturing methylated nucleotides.

[0009] In some aspects, the present invention provides methods for evaluating nucleic acids. Exemplary methods include providing a sample comprising methylated nucleic acids; contacting the sample with a polypeptide comprising a methyl-CpG-binding domain (MBD); and capturing the methylated nucleic acids with the polypeptide to separate the methylated nucleic acids. In some aspects, the methylated nucleic acids are cell-free DNA (cfDNA). The cfDNA can be circulating tumor DNA (ctDNA).

[0010] In some aspects, the polypeptide comprises a plurality of MBDs. In some aspects, the polypeptide comprises a capture tag or is associated with a capture tag. In some aspects, the polypeptide is attached to a solid surface via the capture tag. In some aspects, the polypeptide is attached to the solid surface after capturing the methylated DNA. In some aspects, the polypeptide is attached to the solid surface before capturing the methylated DNA. In some aspects, the capture tag is cleavable. In such methods, the polypeptide with the captured methylated nucleic acid can be released from the solid surface by cleavage of the capture tag.

[0011] Exemplary capture tags include protein A, protein G, streptavidin, cell surface vimentin (CSV), human prostate specific membrane antigen (PSMA), antibodies, antibody fragments, IgG Fc heavy chain, viral proteins, chitin binding domains, maltose binding protein, protein L'B' repeats, oligonucleotides and other capture tags known in the art. Capture tags can be captured by homologous moieties attached to a solid surface. The solid surface can include, for example, beads, flow cells or columns.

[0012] In certain aspects, the MBD is from or derived from MBD2. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The structure of the protein termed "prod-aqg" is shown.

[0014] Figure 2 The structure of the protein termed "prod-bqi" is shown.

[0015] Figure 3 The structure of the protein termed "prod-arj" is shown.

[0016] Figure 4 is a gel showing the production of a protein called "prod-aqg" in E. coli.

[0017] Figure 5 is a gel showing the production of a protein called "prod-bqi" in E. coli.

[0018] Figure 6 is a gel showing the production of a protein called "prod-arj" in E. coli. DETAILED DESCRIPTION

[0019] The present invention provides methods and compositions for capturing and separating methylated DNA from a sample using a polypeptide comprising a methyl-CpG-binding domain. In a preferred aspect, the captured DNA is cell-free DNA (cfDNA), such as circulating tumor DNA (ctDNA). ctDNA is an important biomarker for assessing and diagnosing cancer and is easily identified from other cfDNA due to its methylation state. In addition, the methylation profile of ctDNA (including any changes) can be used to detect, classify and monitor the progress of cancer or its treatment.

[0020] In some aspects, the present invention provides methods for evaluating nucleic acids. Exemplary methods include providing a sample comprising methylated nucleic acids; contacting the sample with a polypeptide comprising a methyl-CpG-binding domain (MBD); and capturing the methylated nucleic acids with the polypeptide to separate the methylated nucleic acids. In some aspects, the methylated nucleic acids are cell-free DNA (cfDNA). The cfDNA can be circulating tumor DNA (ctDNA).

[0021] In certain aspects, the polypeptide comprises a plurality of BMDs. The plurality of BMDs can be used to simultaneously capture a plurality of methylated nucleic acids and / or to capture a plurality of methylated bases of a single methylated nucleic acid.

[0022] The naturally occurring methyl-CpG binding domain protein 2 (MBD2) has the ability to specifically bind to 5mC, especially when 5mC is present in CpG islands. However, the present inventors have discovered that a large number of polypeptides comprising a methyl-CpG-binding domain (including polypeptides engineered from the naturally occurring MBD2 sequence) are capable of capturing methylated nucleotides.

[0023] In some aspects, the polypeptide comprises a capture tag or is associated with a capture tag. In some aspects, the polypeptide is attached to a solid surface via the capture tag. In some aspects, the polypeptide is attached to the solid surface after capturing the methylated DNA. In some aspects, the polypeptide is attached to the solid surface before capturing the methylated DNA. In some aspects, the capture tag is cleavable. In such methods, the polypeptide with the captured methylated nucleic acid can be released from the solid surface by cleavage of the capture tag.

[0024] Table 1 below provides exemplary capture tags and cognate ligands attached to exemplary types of solid supports.

[0025]

[0026]

[0027] Figure 1-3 Schematic diagrams of exemplary polypeptides of the invention are provided.

[0028] Figure 1 A schematic diagram of an exemplary polypeptide "prod-aqg" of the present invention is shown. The polypeptide is a fusion protein including an MBD. The MBD can be from or derived from methyl-CpG binding domain protein 2 (MBD2). However, the inventors have found that MBDs of different polypeptide sequences easily capture methylated nucleic acids. The fusion protein also includes a capture tag, in this case a polyhistidine (His tag), which can be used to attach the fusion protein to a solid surface (e.g., beads, columns, or flow cells). The His tag can be used, for example, to attach to a functionalized or nickel surface. Prior to capturing methylated nucleic acids, the polypeptide can be attached to a solid surface via a tag. Alternatively, the polypeptide can capture methylated nucleic acids and then be attached to a solid surface via a tag to separate the captured product.

[0029] Also like Figure 1 As shown, the capture tag can be attached to the polypeptide / fusion protein by a cleavable part or linker (in this case, an Avi tag), which can release the polypeptide methylated nucleic acid from the solid surface. In addition, the polypeptide / fusion protein can further comprise additional capture tags or other functional parts.

[0030] Also like Figure 1 As shown, the polypeptide / fusion protein may further comprise an enzyme, such as a ligase, a polymerase, a transferase, etc. Figure 1 As shown, the enzyme is the ligase BirA. Advantageously, BirA (bifunctional biotin-[acetyl-CoA carboxylase] holoenzyme synthetase / DNA binding transcriptional repressor, biotinyl-5'-AMP binding) acts as both a ligase and a transcriptional repressor of the biotin operon. Biotin can be used, for example, to capture polypeptide-methylated nucleic acids through biotin-streptavidin interactions.

[0031] In an exemplary method, Prod-Aqg is introduced with Ni2 beads and bound to the beads via the His tag. The beads are then introduced into a sample of sheared DNA and incubated. After incubation, the beads are washed, preferably in a low salt wash solution. The bead-bound MBD is eluted in a salt-based eluent.

[0032] Figure 2 A schematic diagram of an exemplary polypeptide "prod-bqi" of the invention is provided. In contrast to the "prod-aqg" polypeptide, this polypeptide comprises only a single MBD.

[0033] Figure 3 A schematic diagram of an exemplary polypeptide "prod-arj" of the invention is provided.

[0034] Exemplary capture tags include protein A, protein G, streptavidin, cell surface vimentin (CSV), human prostate specific membrane antigen (PSMA), antibodies, antibody fragments, IgG Fc heavy chain, viral proteins, chitin binding domains, maltose binding protein, protein L'B' repeats, oligonucleotides and other capture tags known in the art. Capture tags can be captured by homologous moieties attached to a solid surface. The solid surface can include, for example, beads, flow cells or columns.

[0035] In certain aspects, the MBD is from or derived from MBD2.

[0036] In some aspects, the captured methylated nucleic acid is sequenced or used in another downstream application. In some aspects, the methylation state of the nucleic acid is assessed. In some aspects, the captured methylated nucleic acid is ctDNA. In some aspects, the method of the present invention is used to diagnose or predict cancer.

[0037] Example

[0038] Example 1

[0039] MBD is a well-characterized domain of many cytosine-specific methyltransferase proteins. In this example, the MBD is together with other entities that can be rapidly 'pulled down', i.e. MBD2-6xHis can be pulled down by nickel beads. This allows MBD-dependent enrichment of 5mc-containing oligonucleotides from sheared DNA sample pre-library preparation.

[0040] 1uL of 100ng / uL pD441 expression plasmid encoding polypeptide prod-aqg, prod-bqi or prod-arj was transformed into 30uL NEBExpress cells, 100uL recovered for 20 minutes and inoculated into LB / Kan plates. LB / Kan starter cultures were started from a single colony and grown overnight at 30°C.

[0041] Use the starter culture (OD600 below) to inoculate a single 55mL TB / Kan culture at a ratio of 1:100. 100uL starter culture is mixed with 400uL 50% glycerol and stored at -80°C for working cell stock. The culture is grown at 37°C 250rpm until OD600 is 1.0 (about 3 hours). When OD600 is 1.0, the culture is then divided into 2×25mL, reaches the appropriate temperature (37°C and 24°C), and induces under 1mM IPTG. Collect 1mL IPTG pre-sample before induction, and precipitate.

[0042] The expression cultures were grown for another 5 hours at the specified temperature. 1 mL of the culture pellet (20 k x g, 5 minutes, 4 ° C) was used for soluble protein expression. The expression cultures were grown overnight at 16 ° C. All cultures were grown in 250 mL erlenmeyer flasks with shaking at 250 rpm. Final 16 ° C OD600s: 1 mL of the culture pellet (20 k x g, 5 minutes, 4 ° C) was used for soluble protein expression.

[0043] The pellet was lysed to obtain the supernatant for analysis of soluble protein expression by SDS page, the lysis step included resuspending the pellet in (OD600*100) uL of 400 mM lysis buffer, saving 50 uL of the 'total' sample, and then lysing the beads. 50 uL of soluble material was saved.

[0044] SDS-PAGE: 30uL of the above sample + 20uL 4X loading buffer. The sample was treated at 90℃ for 5 minutes, loaded into Tris-MES buffer containing 4-20% GenScript gel, and run on the gel at 160V for 45 minutes. The results are presented in Figure 4 In the leftmost gel image.

[0045] MBD fusion constructs were found to be soluble for the most part simply under 37°C growth and 37°C expression period. 2 L of each fusion protein:

[0046] prod-aqg (N-terminal his tag, Avi tag, MBD2_143-220, GlySer linker, MBD2_143-220, BirA substrate peptide) 24.25 kDa, pI about 9.63

[0047] prod-bqi (N-terminal his tag, Avi tag, MBD2_143-220, GlySer linker, BirA substrate peptide) 15.6 kDa, pI about 9.0

[0048] prod-arj (N-terminal his tag, MBD2_143-220x2) 20 kDa, pI about 9.86

[0049] Starter cultures from cell stocks generated in Example 1 were grown at 30°C. Cultures were grown at 37°C until OD600 was between 1.0 and 2.0. Induction with 1 mM IPTG. Expression at 37°C for 4-5 hours.

[0050] For each, 4 x 500 mL TB / Kan were inoculated in a 2 L Erlenmeyer flask. The culture was induced with a final concentration of 1 mM IPTG and grown for another 4 hours at 37°C, 250 rpm. The cells were pelleted (5 krpm, 4°C) for 45 minutes. The cell pellets were stored at -20°C.

[0051] Cell paste treatment: First, 8.5 g of the pellet was resuspended in 46 mL of nickel buffer A (15 mM Tris-HCl pH 7.5, 400 mM NaCl, 20 mM imidazole, 10% glycerol). Then, lysis was performed by ultrasonic treatment at 60% power; 30 seconds on and 30 seconds off, 20 cycles. 60 mL of whole cell lysate was brought to a PEI concentration of 0.3% by adding 1.85 mL of 9.7% PEI stock solution. The PEI / lysate mixture was incubated on ice at 4°C for 1 hour. The PEI pellet was discarded and the supernatant was transferred to 35% AmSO4. The pellet in 35% AmSO4 was discarded and then transferred to 70%. Incubate at 4°C for 30 minutes and centrifuge to precipitate insoluble material. The pellet was retained and stored at -20 until use.

[0052] Nickel Column: Resuspend the pellet in 5x Cell Disruption Nickel Buffer A (50 mL). Load onto 3×5 mL freshly nickel-charged capto-chelated HiTrap columns (3 mL / min). Then wash with 5 CV 2M nickel salt buffer. Run (3 mL / min). Elute 0-75% B over 10 CV and capture 1.5 mL fractions. (3 mL / min). Separate fractions on Tris-MES 4-20% SDS-PAGE gel. The separated polypeptide forms a band at 30 kDa.

[0053] Heparin column: Fractions D6 to G2 from the nickel eluate were combined (approximately 50 mL) and diluted to approximately 200 mM NaCl with universal dilution buffer. The fractions were then loaded onto 3×5 mL Capto Heparin Impres Hi-Traps at 3 mL / min. The fractions were then eluted with 0-75% buffer B over 8 CV.

[0054] A corresponding amount of material flows through the heparin column and produces small and large peaks.

[0055] The fractions were run on SDS-PAGE; using Tris-MOPS and using a 4-12% gel the loading dye [DTT] was increased by 10 mM to see if the protein migrated in the same way under different buffer conditions (Gel 2). 160 V, 35 minutes. It was found that using this column provided clearer results and provided a separated band representing the 30 kDa polypeptide.

[0056] Fractions D8 to E2 (approximately 12 mL at 4.5 mg / mL, approximately 47.5 mS / cm) were combined and mixed. 6 mL of this combination was added to 6 mL 100% glycerol. Result: 12 mL at approximately 2.25 mg / mL in approximately 10 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.1 mM EDTA, 50% glycerol.

[0057] SEC: The remaining pool was concentrated by centrifugation (Amicron 10 kDa MWCO, 1.7 mL) from about 6 mL to 500 uL and applied to a Superdex 200 plus 15 / 300 size exclusion column in 350 mM NaCl buffer at 0.25 mL / min. A 500 uL fraction was captured.

[0058] Running on an SDS-PAGE gel appeared to reveal a pure product that oligomerized in a stable manner under these SDS conditions.

[0059] The previous samples were at about 350 mM NaCl. In subsequent replicates, the salt level was increased.

[0060] Cell paste treatment: First, 8.0 g of the pellet was resuspended in 46 mL of nickel buffer A (15 mM Tris-HCl pH 7.5, 400 mM NaCl, 20 mM imidazole, 10% glycerol). Lysis was performed by sonication. 60% power; 30 seconds on, 30 seconds off, 20 cycles. 60 mL of whole cell lysate was made 0.3% PEI by adding 1.85 mL of 9.7% PEI stock solution. The PEI / lysate mixture was incubated on ice at 4°C for 1 hour. The PEI pellet was discarded and sup (45 mL) was brought to 70% AmSO4 with 22.39 g AmSO4. The sample was vortexed frequently and incubated at 4°C for 30 minutes. Insoluble material was precipitated at 15 krpm at 4°C for 45 minutes. The pellet was retained and stored at -20°C until use.

[0061] Nickel Column: Resuspend each pellet in 5x Cell Disruption Nickel Buffer A (50 mL). Load onto 3 x 5 mL freshly nickel-charged capto-chelated HiTrap columns (3 mL / min). Wash columns with 1M NaOH / 200 mM Tris / dH2O between runs. Wash with 5CV 2M nickel buffer. (3.2 mL / min). Elute 0-75% B over 10CV. Capture 1.5 mL fractions. (3.2 mL / min). Fractions were exhausted on Tris-MES 4-20% SDS-PAGE gel (Gel 4).

[0062] As expected, both investigated polypeptides were soluble expressed and bound to the nickel resin.

[0063] Heparin column: The eluates are combined and loaded onto a heparin column. It is expected that different amounts of MBD in the peptide will result in different affinities for the heparin column.

[0064] Combine 750uL from each 'aCat177' elution fractions E4 to F10. Combine 750uL with 'aCat179' elution fractions C6 to D12 above. Total = 28.5mL at 400mM NaCl. Dilute this pool with 41.5mL 100mM NaCl buffer to yield approximately 50mL of approximately 222mM NaCl load.

[0065] Example 2

[0066] Several constructs of MBD2 and BirA ligase were generated. Genetic sequences for the following fusion proteins of the invention were generated: prod-aqg: N-terminal his tag, Avi tag, MBD2_143-220, GlySer linker, MBD2_143-220, BirA substrate peptide; aCat179: N-terminal his tag, Avi tag, MBD2_143-220, GlySer linker, BirA substrate peptide; prod-pqi: N-terminal his tag, MBD2_143-220, GlySer linker; and prod-arj: BirA ligase. All gene designs were placed in pD441 vector backbone (strong RBS, T5 promoter) and pD1841 (strong RBS, phosphate starvation promoter).

[0067] like Figure 4 As shown, all of these fusion proteins were successfully expressed and tested using E. coli.

[0068] >BirA ligase, prod-aqh (SEQ ID NO: 1)

[0069] MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGY

[0070] SLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRR

[0071] GRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDL

[0072] YLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRN

[0073] TLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGA

[0074] LLLEQDGIIKPWMGGEISLRSAEK

[0075] >MBD2, prod-aqg (SEQ ID NO: 2)

[0076] MHHHHHHAGGLNDIFEAQKIEWHEDTGGSATESGKRMDCPALPPGWKKEEVIRKSGLS

[0077] AGKSDVYYFSPSGKKFRSKPQLARYLGNTVDLSSFDFRTGKMMPSKLQKDDDKEFGGG

[0078] GSGGGGSGGGGATESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVDLSSFDFRTGKMMPSKLQKLHHILDAQKMVWNHRG*

[0079] >MBD2, prod-bqi (SEQ ID NO:3)

[0080] MHHHHHHGAGGLNDIFEAQKIEWHEDTGGSATESGKRMDCPALPPGWKKEEVIRKSGL

[0081] SAGKSDVYYFSPSGKKFRSKPQLARYLGNTVDLSSFDFRTGKMMPSKLQKDDDKEFGGGGSGGGGSGGGGLHHILDAQKMVWNHRG*

[0082] >MBD2_prod-arj (SEQ ID NO:4)

[0083] MHHHHHHATESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQL

[0084] ARYLGNTVDLSSFDFRTGKMMPSKLQKDDDKEFGGGGSGGGGSGGGGATESGKRMDC

[0085] PALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVDLSSFDFRTGKMMPSKLQKG*

[0086] >MDB2-isotype-alternative 1 (SEQ ID NO:5)

[0087] MRAHPGGGRC CPEQEEGESA AGGSGAGGDS AIEQGGQGSA LAPSPVSGVR REGARGGGRGRGRWKQAGRG GGVCGRGRGR GRGRGRGRGR GRGRGRPPSG GSGLGGDGGG CGGGGSGGGG APRREPVPFPSGSAGPGPRG PRATESGKRM DCPALPPGWK KEEVIRKSGL SAGKSDVYYF SPSGKKFRSK PQLARYLGNTVDLSSFDFRT GKMMPSKLQK NKQRLRNDPL NQNKGKPDLN TTLPIRQTAS IFKQPVTKVT NHPSNKVKSDPQRMNEQPRQ LFWEKRLQGL SASDVTEQII KTMELPKGLQ GVGPGSNDET LLSAVASALH TSSAPITGQVSAAVEKNPAV WLNTSQPLCK AFIVTDEDIR KQEERVQQVR KKLEEALMAD ILSRAADTEE MDIEMDSGDEA

[0088] >MDB2 - Isotype - Alternative 2 (SEQ ID NO:6)

[0089] MRAHPGGGRC CPEQEEGESA AGGSGAGGDS AIEQGGQGSA LAPSPVSGVR REGARGGGRGRGRWKQAGRG GGVCGRGRGR GRGRGRGRGR GRGRGRPPSG GSGLGGDGGG CGGGGSGGGG APRREPVPFPSGSAGPGPRG PRATESGKRM DCPALPPGWK KEEVIRKSGL SAGKSDVYYF SPSGKKFRSK PQLARYLGNTVDLSSFDFRT GKMMPSKLQK NKQRLRNDPL NQNKFVCTFL L

Claims

1. A method for evaluating a nucleic acid, the method comprising: providing a sample comprising methylated nucleic acid; contacting the sample with a polypeptide comprising a methyl-CpG-binding domain (MBD); as well as The methylated nucleic acid is captured with the polypeptide to separate the methylated nucleic acid.

2. The method of claim 1, wherein the nucleic acid is cfDNA.

3. The method of claim 2, wherein the cfDNA is ctDNA.

4. The method of claim 1, wherein the polypeptide comprises a plurality of said MBDs.

5. The method of claim 1, wherein the polypeptide comprises a capture tag or is attached to the capture tag. The method of claim 5 , wherein the polypeptide is attached to a solid surface via the capture tag. The method according to claim 6 , wherein the method further comprises a separation step of washing away non-captured nucleic acids.

8. The method of claim 6, wherein the solid surface is a bead, a column, or a flow cell.

9. The method of claim 8, wherein the solid surface comprises nickel and the capture tag is a His tag.

10. The method of claim 5, wherein the capture tag comprises one or more of the following: protein A, protein G, streptavidin, cell surface vimentin (CSV), human prostate specific membrane antigen (PSMA), antibodies, antibody fragments, IgG Fc heavy chain, viral proteins, chitin binding domains, maltose binding protein, protein L'B' repeats, and oligonucleotides.

11. The method of claim 1, wherein the MBD is from or derived from MBD2.

12. The method of claim 1 further comprising a separation step, optionally wherein the separation step comprises capturing on nickel and washing away non-captured material.

13. The method of claim 1, further comprising diagnosing the subject as having cancer based on the characteristics and / or methylation status of the captured methylated nucleotides.

14. The method of claim 12, wherein the method is used to diagnose a subject as having minimal residual disease after receiving cancer treatment.

15. The method of claim 1, further comprising expressing the polypeptide in Escherichia coli using an expression vector.

16. The method of claim 1, wherein the methylated nucleic acid is less than about 200 bp in length. The method according to claim 15 , wherein the methylated nucleic acid is derived from fragmented nucleic acid.

18. The method of claim 1, further comprising sequencing the captured methylated nucleic acids.

19. The method of claim 1, wherein the polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:

6.

20. The method of claim 1, wherein the polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:

4.

21. The method of claim 1, wherein the polypeptide comprises SEQ ID NO:2 or SEQ ID NO:3.

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