Capture of methylated nucleic acids

Abstract

The present invention provides a method and composition for capturing and isolating methylated DNA from a sample using a polypeptide that comprises methyl-CpG binding domain. The captured DNA is cell-free DNA (cfDNA), such as circulating tumor DNA (ctDNA). cfDNA is an important biomarker for evaluating and diagnosing cancer, and is easily identified from other cfDNA due to its methylation status. Furthermore, the methylation profile of ctDNA (including any changes) can be used to detect, classify and monitor the progression of cancer or its treatment.

Description

[Technical field]

[0001] Sequence Listing A "Sequence Listing XML" has been submitted herewith in XML file format, and (i) has a file name of WMG-010-01WO.xml; (ii) has a creation date of March 16, 2023; and (iii) has a file size of 7,481 bytes, and the data in the XML file is incorporated by reference.

[0002] Technical Field The present disclosure relates generally to methods and compositions for capturing methylated nucleic acids using capture moieties that include a methyl-CpG binding domain. [Background technology]

[0003] background Studies have shown that epigenetics can play a crucial role in disease. One of the more commonly examined epigenetic modifications is DNA methylation. DNA methylation is believed to help regulate gene expression. Thus, many diseases (e.g., cancer) are associated with increased levels of DNA methylation. In cells, most DNA methylation occurs via the addition of a methyl group (5mC) to the 5' carbon on cytosine residues, especially those found in stretches of CpG dinucleotides (CpG islands). It is believed that in certain circumstances, increased methylation (hypermethylation) is a natural response to certain conditions (e.g., cancer) and helps reduce the transcription of certain genes. In cells, methylated DNA can reduce transcription by preventing the binding of certain transcription factors and / or recruit repressor complexes to genes.

[0004] Due to its emerging role in cancer, it is becoming increasingly important to evaluate the methylation status of genetic material derived from cancer cells (e.g., tumor cells). Cell-free DNA (cfDNA) derived from cancer cells (e.g., circulating tumor DNA (ctDNA)) has a completely different methylation status compared to other cfDNA. Thus, it represents a promising tool for cancer diagnosis and testing. Summary of the Invention [Means for solving the problem]

[0005] Abstract The present invention provides a method and composition for capturing and isolating methylated DNA from a sample using a polypeptide that comprises methyl-CpG binding domain.In a preferred aspect, the captured DNA is cell-free DNA (cfDNA), such as circulating tumor DNA (ctDNA).cfDNA is an important biomarker for evaluating and diagnosing cancer, and is easily identified from other cfDNA due to its methylation status.In addition, the methylation profile of ctDNA (including any changes) can be used to detect, classify and monitor the progression of cancer or its treatment.

[0006] Although methods exist for capturing and isolating methylated DNA, these prior methods (e.g., methylation immunoprecipitation coupled with next-generation sequencing in a process known as methylated DNA immunoprecipitation sequencing (MeDIP-seq)) require high concentrations of DNA and / or are unable to capture and isolate short nucleic acids. However, ctDNA is generally short in length (usually less than 200 bp) and found at low concentrations. The disclosed methods and compositions using polypeptides containing methyl-CpG binding domains (MBDs) can capture short methylated DNA sequences found at low concentrations.

[0007] Naturally occurring methyl-CpG binding domain protein 2 (MBD2) has the ability to specifically bind 5mC, especially when found in CpG islands. However, the inventors have discovered that a wide range of polypeptide methyl-CpG binding domains, including those engineered from the naturally occurring MBD2 sequence, can capture methylated nucleotides.

[0008] In a particular aspect, the present invention provides a method for evaluating nucleic acid.An exemplary method includes the steps of: providing a sample that contains methylated nucleic acid; contacting the sample with a polypeptide that contains methyl-CpG binding domain (MBD); and capturing the methylated nucleic acid with the polypeptide to isolate the methylated nucleic acid.In a particular aspect, the methylated nucleic acid is cell-free DNA (cfDNA).The cfDNA can be circulating tumor DNA (ctDNA).

[0009] In certain aspects, the polypeptide comprises multiple MBDs. In certain aspects, the pol peptide comprises or is attached to a capture tag. In certain aspects, the pol peptide is attached to a solid surface via the capture tag. In certain aspects, the polypeptide is attached to the solid surface after capturing the methylated DNA. In certain aspects, the polypeptide is attached to the solid surface before capturing the methylated DNA. In certain aspects, the capture tag is cleavable. In such methods, the polypeptide with the captured methylated nucleic acid can be released from the solid surface via cleavage of the capture tag.

[0010] 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 cognate moieties attached to a solid surface. The solid surface can include, for example, beads, flow cells, or columns.

[0011] In certain aspects, the MBD is derived or obtained from MBD2. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the structure of the protein called "prod-aqg."

[0013] [Diagram 2] FIG. 2 shows the structure of a protein called "prod-bqi."

[0014] [Diagram 3] FIG. 3 shows the structure of a protein called "prod-arj."

[0015] [Figure 4] FIG. 4 is a gel showing that a protein called "prod-aqg" was produced by E. coli.

[0016] [Diagram 5] FIG. 5 is a gel showing that a protein called "prod-bqi" was produced by E. coli.

[0017] [Figure 6] FIG. 6 is a gel showing that a protein called "prod-arj" was produced by E. coli. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description The present invention provides a method and composition for capturing and isolating methylated DNA from a sample using a polypeptide that comprises methyl-CpG binding domain.In a preferred aspect, the captured DNA is cell-free DNA (cfDNA), such as circulating tumor DNA (ctDNA).cfDNA is an important biomarker for evaluating and diagnosing cancer, and is easily identified from other cfDNA due to its methylation status.In addition, the methylation profile of ctDNA (including any changes) can be used to detect, classify and monitor the progression of cancer or its treatment.

[0019] In a particular aspect, the present invention provides a method for evaluating nucleic acid.An exemplary method includes the steps of: providing a sample that contains methylated nucleic acid; contacting the sample with a polypeptide that contains methyl-CpG binding domain (MBD); and capturing the methylated nucleic acid with the polypeptide to isolate the methylated nucleic acid.In a particular aspect, the methylated nucleic acid is cell-free DNA (cfDNA).The cfDNA can be circulating tumor DNA (ctDNA).

[0020] In certain aspects, the polypeptide comprises multiple BMDs, which can be used to simultaneously capture multiple methylated nucleic acids and / or capture multiple methylated bases in a single methylated nucleic acid.

[0021] The naturally occurring methyl-CpG binding domain protein 2 (MBD2) has the ability to specifically bind 5mC, particularly when found in CpG islands. However, the inventors have discovered that a wide range of polypeptide methyl-CpG binding domains, including those engineered from the naturally occurring MBD2 sequence, can capture methylated nucleotides.

[0022] In certain aspects, the polypeptide comprises or is attached to a capture tag. In certain aspects, the polypeptide is attached to a solid surface via the capture tag. In certain aspects, the polypeptide is attached to the solid surface after capturing the methylated DNA. In certain aspects, the polypeptide is attached to the solid surface before capturing the methylated DNA. In certain aspects, the capture tag is cleavable. In such methods, the polypeptide with the captured methylated nucleic acid can be released from the solid surface via cleavage of the capture tag.

[0023] Table 1 below provides exemplary capture tags and their cognate ligands attached to exemplary types of solid supports. [Table 1]

[0024] 1-3 provide schematic diagrams of exemplary polypeptides of the invention.

[0025] FIG. 1 shows a schematic diagram of an exemplary polypeptide "prod-aqg" of the invention. The polypeptide is a fusion protein that includes an MBD. The MBD is derived from or obtained from methyl-CpG binding domain protein 2 (MBD2). However, the inventors have discovered that MBDs of different polypeptide sequences readily 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., a bead, a column, or a flow cell). The His tag can be used, for example, for attachment to a functionalized or nickel surface. The polypeptide can be attached to a solid surface via the tag prior to capturing the methylated nucleic acid. Alternatively, the polypeptide can capture the methylated nucleic acid and then be attached to a solid surface via the tag to isolate the capture product.

[0026] As also shown in Figure 1, the capture tag can be attached to the polypeptide / fusion protein via a cleavable moiety or linker (in this case, an Avi Tag) that can release the polypeptide-methylated nucleic acid from the solid surface. Additionally, the polypeptide / fusion protein can further comprise an additional capture tag or functional portion.

[0027] As also shown in Figure 1, the polypeptide / fusion protein may further comprise an enzyme (e.g., ligase, polymerase, transferase, etc.). As shown in Figure 1, the enzyme is the ligase BirA. Advantageously, BirA (bifunctional biotin-[acetyl-CoA carboxylase] holoenzyme synthetase / DNA-binding transcriptional repressor, biotin-5'-AMP binding) acts both as a ligase and a transcriptional repressor of the biotin operon. Biotin can be used, for example, to capture the polypeptide-methylated nucleic acid via biotin-streptavidin interaction.

[0028] 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 to a sample of sheared DNA and incubated. After incubation, the beads are preferably washed in a low salt wash. The MBD bound to the beads is eluted in a salt-based elution.

[0029] Figure 2 provides a schematic diagram of an exemplary polypeptide of the invention, "prod-bqi." In contrast to the "prod-aqg" polypeptide, this polypeptide contains only one MBD.

[0030] FIG. 3 provides a schematic diagram of an exemplary polypeptide of the invention, "prod-arj."

[0031] 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. The capture tag may be captured by a cognate moiety attached to a solid surface. The solid surface may include, for example, a bead, a flow cell, or a column.

[0032] In certain aspects, the MBD is derived or obtained from MBD2.

[0033] In certain aspects, the captured methylated nucleic acid can be sequenced or used in another downstream application. In certain aspects, the methylation status of the nucleic acid is evaluated. In certain aspects, the captured methylated nucleic acid is ctDNA. In certain aspects, the method of the present invention is used to diagnose or prognose cancer. EXAMPLES

[0034] Working Example Example 1 The MBD is a well-characterized domain of many cytosine-specific methyltransferase proteins. In this example, the MBD is joined to another entity that can be rapidly "pulled down", i.e., MBD2-6xHis can be pulled down by nickel beads. This allows for MBD-dependent enrichment of 5mc-containing oligos from sheared DNA sample pre-library preparations.

[0035] pD441 expression plasmids encoding polypeptides prod-aqg, prod-bqi, or prod-arj, 1 μL 100 ng / μL, were transformed into 30 μL NEBExpress cells and 100 μL 20 min recovered on LB / Kan. L LB / Kan starter cultures were initiated from single colonies and grown overnight at 30°C.

[0036] The starter culture (OD600 below) was used to inoculate a single 55mL TB / Kan culture at 1:100. 100μL starter culture was mixed with 400μL 50% glycerol and set aside at -80°C for working cell stock. The culture was grown at 37°C, 250 rpm until the OD600 was 1.0 (approximately 3 hours). At OD600 1.0, the culture was then split into 2x25mL, brought to the appropriate temperature (37°C vs 24°C) and induced with 1mM IPTG. A 1mL pre-IPTG sample was taken prior to induction and pelleted.

[0037] Expression cultures were grown for an additional 5 hours at the indicated temperatures. 1 mL of culture was pelleted at 20k x g for 5 minutes at 4°C for soluble protein expression. 16°C Expression cultures were grown overnight. All cultures were grown in 250 mL Erlenmeyer flasks with shaking at 250 rpm. Final 16°C OD600s: 1 mL of culture was pelleted at 20k x g for 5 minutes at 4°C for soluble protein expression.

[0038] Dissolve the pellet to obtain the above and analyze soluble protein expression by SDS page. This involves resuspending the pellet in (OD600*100) μL of 400mM lysis buffer, setting aside 50 μL of "total" sample, then bead lysis. Set aside 50 μL of soluble material.

[0039] SDS-PAGE: 30 μL of the above sample + 20 μL 4× loading buffer (90° C., 5 min) was loaded onto a 4-20% GenScript Gel in Tris-MES buffer and run on the gel for 45 min at 160 V. The results are shown in the leftmost gel image of FIG.

[0040] The MBD fusion constructs were found to be largely soluble only at 37°C growth and expression periods. prod-aqg (N-terminal his tag, AviTag, MBD2_143-220, GlySer linker, MBD2_143-220, BirA substrate peptide) 24.25 kDa, pI approx. 9.63 prod-bqi (N-terminal his tag, AviTag, MBD2_143-220, GlySer linker, BirA substrate peptide) 15.6 kDa, pI approx. 9.0 prod-arj (N-terminal his tag, MBD2_143-220×2) 20 kDa, pI approx. 9.86

[0041] A 30° C. starter culture was grown from the cell stock generated in Example 1. The culture was grown at 37° C. to an OD600 between 1.0-2.0, induced with 1 mM IPTG, and expressed at 37° C. for 4-5 hours.

[0042] For each, 4 x 500 mL TB / Kan were inoculated into 2 L Erlenmeyer flasks. Cultures were induced with a final concentration of 1 mM IPTG and grown for an additional 4 hours at 37°C, 250 rpm. Cells were pelleted for 45 minutes at 5 krpm at 4°C. Cell pellets were stored at -20°C.

[0043] Cell paste processing: 8.5g pellet was first resuspended in 46mL of Nickel Buffer A (15mM Tris-HCl pH 7.5, 400mM NaCl, 20mM Imidazole, 10% glycerol). Then lysed via sonication at 60% output; 30s on 30s off 20 cycles. 60mL total cell lysate was made 0.3% PEI by adding 1.85mL 9.7% PEI stock. The PEI / lysate mix was incubated on ice at 4°C for 1 hour. The PEI pellet was discarded, suspended in 35% AmSO, the 35% AmSO4 pellet was discarded, then made 70%. Incubated at 4°C for 30 minutes and insoluble material was centrifuged. The pellet was kept and stored at -20°C until use.

[0044] Nickel column: The pellet was resuspended in 5x Cell Disruption Nickel Buffer A (50 mL). It was loaded onto 3x5 mL freshly nickel-loaded capto chelating HiTrap columns (3 mL / min). This was followed by a 5 CV 2M salt Nickel buffer wash (3 mL / min). Elution was 0-75% B over 10 CV, giving 1.5 mL fractions (3 mL / min). Fractions were run on a Tris-MES 4-20% SDS-PAGE gel. The isolated polypeptide banded on the gel at 30 kDa.

[0045] Heparin column: Fractions D6-G2 from the nickel elution were pooled (approximately 50 mL) and diluted to approximately 200 mM NaCl with universal dilution buffer. The fractions were then loaded onto 3 x 5 mL Capto Heparin Impres Hi-Traps at 3 mL / min. They were then eluted with 0-75% Buffer B over 8 CV.

[0046] As a result, a lot of material flowed through the heparin column, giving rise to a minor and a major peak.

[0047] Fractions were run on SDS-PAGE; increasing dye loading [DTT] in 10 mM increments, using Tris-MOPS, and a 4-12% gel to determine if the protein migrated similarly under different buffer conditions (gel 2). 160V, 35 min. Use of this column was found to provide a cleaner result and an isolated band at 30 kDa representing the polypeptide.

[0048] Fractions D8-E2 (approximately 12 mL at 4.5 mg / mL, approximately 47.5 mS / cm) were pooled and mixed. 6 mL of this pool 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.

[0049] SEC: The remaining pool was concentrated from approximately 6 mL to 500 μL by centrifugation (Amicron 10 kDa MWCO, 1.7 mL) and applied to a Superdex 200 increase 15 / 300 size exclusion chromatography column at 0.25 mL / min in 350 mM NaCl buffer. 500 μL fractions were obtained.

[0050] Runs on SDS-PAGE gels appeared to show pure products that oligomerized in a stable manner under these SDS conditions.

[0051] The previous sample was in approximately 350 mM NaCl. In subsequent iterations, the salt level was increased.

[0052] Cell paste processing: 8.0 g pellet was first resuspended in 46 mL of Nickel Buffer A (15 mM Tris-HCl pH 7.5, 400 mM NaCl, 20 mM imidazole, 10% glycerol). Lysis via sonication. 60% power; 30 sec on, 30 sec off, 20 cycles. 60 mL total cell lysate was made 0.3% PEI by adding 1.85 mL 9.7% PEI stock. The PEI / lysate mix was incubated on ice at 4°C for 1 hour. The PEI pellet was discarded and the supernatant (45 mL) was made 70% AmSO4 with 22.39 g AmSO4. The sample was vortexed well and incubated at 4°C for 30 minutes. Insoluble material was pelleted for 45 minutes at 15 krpm at 4°C. The pellet was retained and stored at -20°C until use.

[0053] Nickel column: Each pellet was resuspended in 5x Cell disruption Nickel buffer A (50 mL). Loaded onto 3x5 mL freshly nickel-loaded capto chelating HiTrap columns (3 mL / min). Columns were washed with 1 M NaOH / 200 mM Tris / dH2O between runs. Washed with 5 CV 2M salt Nickel buffer (3.2 mL / min). Elute 0-75% B over 10 CV. Obtained 1.5 mL fractions (3.2 mL / min). Fractions were run on a Tris-MES 4-20% SDS-PAGE gel (Gel 4).

[0054] Both tested polypeptides were solubly expressed and, as intended, attached to nickel resin.

[0055] Heparin column: The eluates were pooled and loaded onto a heparin column, it being understood that the different numbers of MBDs in the polypeptides confer different affinities to the heparin column.

[0056] Pool 750 μL from each "aCat177" elution fraction E4-F10. Pool 750 μL with the above pool of "aCat179" elution fractions C6-D12. Total = 28.5 mL with 40 mM NaCl. Dilute this pool with 41.5 mL 100 mM NaCl buffer to yield approximately 50 mL of approximately 222 mM NaCl load.

[0057] Example 2 Several constructs were generated for MBD2 and BirA ligase. Gene sequences for the following fusion proteins of the invention were generated: prod-aqg: N-terminal his-tag, AviTag, MBD2_143-220, GlySer linker, MBD2_143-220, BirA substrate peptide, aCat179: N-terminal his-tag, AviTag, 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).

[0058] As shown in FIG. 4, these fusion proteins were all successfully expressed and detected using E. coli. >BirA ligase, prod-aqh (SEQ ID NO: 1) [ka] >MBD2, prod-aqg (SEQ ID NO:2) [ka] >MBD2, prod-bqi (SEQ ID NO:3) [ka] >MBD2_prod-arj (SEQ ID NO:4) [ka] >MDB2-isoform-alternative-1 (SEQ ID NO:5) [ka] >MDB2-isoform-alternative-2 (SEQ ID NO:6) [ka]

Claims

1. 1. A method for evaluating a nucleic acid, the method comprising: providing a sample containing methylated nucleic acid; contacting the sample with a polypeptide comprising a methyl-CpG binding domain (MBD); and capturing the methylated nucleic acid with the polypeptide to isolate the methylated nucleic acid; The method includes:

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

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

4. The method of claim 1 , wherein the polypeptide comprises multiple of the MBDs.

5. The method of claim 1 , wherein the polypeptide comprises or is attached to a capture tag.

6. The method of claim 5 , wherein the polypeptide is attached to a solid surface via the capture tag.

7. The method of claim 6 , further comprising a separation step to wash away uncaptured 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. 6. The method of claim 5, wherein the capture tag comprises one or more of protein A, protein G, streptavidin, cell surface vimentin (CSV), human prostate specific membrane antigen (PSMA), an antibody, an antibody fragment, an IgG Fc heavy chain, a viral protein, a chitin binding domain, maltose binding protein, protein L 'B' repeat, and an oligonucleotide.

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

11. 10. The method of claim 1 further comprising a separation step, optionally wherein said separation step comprises washing away captured and uncaptured material on the nickel.

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

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

14. 10. The method of claim 1, further comprising expressing the polypeptide in E. coli using an expression vector.

15. 2. The method of claim 1, wherein the methylated nucleic acid is less than about 200 bp in length.

16. 16. The method of claim 15, wherein the methylated nucleic acid is obtained from a fragmented nucleic acid.

17. The method of claim 1 , further comprising the step of sequencing the captured methylated nucleic acid.

18. 2. 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.

19. 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.

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

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