Patent Application
20170030898
The structure of chromatin plays a significant role in gene expression and development for eukaryotic organisms (Hashimshony et al., 2003). Methylation at the 5 position of the cytosine base, when followed by guanine (CpG) in the promoter region of a protein-coding gene, is an epigenetic modification that has been shown to be involved in DNA condensation and transcriptional inactivation (Wolffe and Matzke, 1999). Aberrant DNA methylation patterns have been implicated in the development of human diseases such as cancer (Feinberg, 2007). Medical research has connected promoter methylation levels for certain genes to therapeutic response in patients. For example, glioma patients with a methylated promoter for the O6-methylguanine-DNA methyltransferase (MGMT) gene exhibit particular sensitivity to alkylating agent chemotherapeutics (Hegi et al., 2005), and breast cancer patients with methylation-dependent silencing of the breast cancer 1, early onset (BRCA1) gene have been shown to have tumors sensitive to cisplatin (Silver et al., 2010). Additionally, physicians can test for epigenetic silencing of the DNA mismatch repair gene MutL homolog 1 (MLHJ) for its prognostic value for patients being treated with colon cancer (Herman et al., 1998, Heyn and Esteller, 2012). Hypermethylation at glutathione S-transferase pi 1 (GSTP1) has also shown promise as a biomarker for diagnosing prostate cancer (Van Neste et al., 2012).
Because promoter methylation has been shown to have predictive, prognostic and diagnostic value, there has been great interest in developing methods for DNA methylation detection with increased sensitivity, specificity, and resolution to increase clinical value (Heyn and Esteller, 2012) and also for discovery purposes to generate reference methylome data (Roadmap Epigenomics et al., 2015).
State of the art methods for DNA methylation detection (whole-genome bisulfite sequencing, reduced representation bisulfite sequencing, CpG specific arrays, and methylation-specific PCR) generally rely on sodium bisulfite conversion of unmethylated cytosine bases to uracil (Heyn and Esteller, 2012). Chemical conversion, however, can degrade more than 90% of the sample DNA (Grunau et al., 2001), and protocols must be assiduously optimized to minimize incomplete deamination of unmethylated cytosine bases and inappropriate conversion of methylated ones to thymine (Genereux et al., 2008). Such errors lead to inaccurate results. Alternatively, immunoprecipitation (IP) based methods such as MeDIP-seq and MBD-seq have been developed. These methods tend to require larger sample inputs (Laird, 2010) and are not capable of providing single methyl CpG site resolution without bisulfite conversion (Pomraning et al., 2009).
To avoid bisulfite conversion while still providing improved resolution, there have been several methods developed recently that use the very methyl binding domain (MBD) proteins involved in forming repressive complexes in vivo to transduce DNA methylation into a signal that can be measured directly (Cipriany et al., 2012, Cipriany et al., 2010, Heimer et al., 2014, Luo et al., 2009, Yu et al., 2010) instead of simply providing sample enrichment as is the case with MBD-seq. These MBD proteins specifically recognize symmetrically methylated CpG dinucleotides in double stranded DNA (Fraga et al., 2003, Hendrich and Bird, 1998, Jorgensen et al., 2006), and therefore, have the potential to enable high resolution DNA methylation detection when paired with sequence specific probe DNA without requiring chemical conversion or sequencing of DNA.
Current MBD-based methods require relatively large amounts of DNA (Heimer, Shatova, Lee, Kaastrup and Sikes, 2014, Luo, Zheng, Wang, Wu, Bai and Lu, 2009, Yu, Blair, Gillespie, Jensen, Myszka, Badran, Ghosh and Chagovetz, 2010) or are not sequence specific (Cipriany, Murphy, Hagarman, Cerf, Latulippe, Levy, Benitez, Tan, Topolancik, Soloway and Craighead, 2012, Cipriany, Zhao, Murphy, Levy, Tan, Craighead and Soloway, 2010). Clinical applications require that both these problems be addressed (Heyn and Esteller, 2012).
Thus there is a need for a very high affinity MBD protein suitable for interfacial use and capable of recognizing a single methylated CpG site. Such a MBD protein will thermodynamically provide a higher fractional coverage of these sites in DNA (Kaastrup et al., 2013), which is particularly important when the total number of sites may be low. Such a reagent would support ongoing research to make methylation analysis on a single DNA molecule (Cipriany, Murphy, Hagarman, Cerf, Latulippe, Levy, Benitez, Tan, Topolancik, Soloway and Craighead, 2012, Cipriany, Zhao, Murphy, Levy, Tan, Craighead and Soloway, 2010, Shapiro et al., 2013, Wang and Bodovitz, 2010) sequence specific.
This present invention provides high affinity variants of human methyl binding domain 2 (hMBD2), and nucleic acids encoding the variants, capable of recognizing and/or binding to methylated DNA. In particular, the hMBD2 variants of the invention recognize and/or bind a DNA sequence with single methylated CpG site with high affinity. The invention provides materials and methods for using the nucleic acid and/or amino acid sequence variants hMBD2 of the invention to detect methylated DNA. The hMBD2 variants of the invention are particularly useful for recognizing and/or binding a DNA sequence with single methylated CpG site with high affinity.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
Provided herein are isolated human methyl bind domain 2 (hMBD2) nucleic acid and amino acid sequence variants. The hMBD2 variants of the invention bind methylated DNA. In particular, the hMBD2 variants of the invention recognize and/or bind DNA comprising a single methylated CpG site, with high affinity. The hMBD2 nucleic acid sequence variants are relative to the reference wild-type hMBD2 sequence (GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAAAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAATTTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTTTCGTACCGGCAAAATG/SEQ ID NO: 16). The hMBD2 amino acid sequence variants are relative to the reference wild-type hMBD2 amino acid sequence (ESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVD LSSFDFRTGKM/SEQ ID NO: 6).
Units, prefixes, and symbols can be denoted in the SI accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.
“About” as used herein means that a number referred to as “about” comprises the recited number plus or minus 1-10% of that recited number. For example, “about” 50 nucleotides can mean 45-55 nucleotides or as few as 49-51 nucleotides depending on the situation. Whenever it appears herein, a numerical range, such as “45-55”, refers to each integer in the given range; e.g., “45-55 nucleotides” means that the nucleic acid can contain 45 nucleotides, 46 nucleotides, etc., up to and including 55 nucleotides.
The terms “oligonucleotide”, “polynucleotide” and “nucleic acid (molecule)” are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may be modified or unmodified and have any three-dimensional structure, and may perform any function, known or unknown. The term “polynucleotide” includes single-, double-stranded and triple helical molecules. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art.
Polynucleotide sequences can be considered to be substantially identical if two molecules hybridize to each other under stringent conditions. However, polynucleotides which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This can occur when a copy of a polynucleotide is created using the maximum codon degeneracy permitted by the genetic code.
As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome (e.g., nucleic acids that encode non-RBM20 proteins). The term “isolated” as used herein with respect to nucleic acids also includes any non-naturally-occurring nucleic acid sequence since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. An isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid.
A “primer” refers to an oligonucleotide containing at least 6 nucleotides, usually single-stranded, that provides a 3′-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis. A “polynucleotide probe” is a polynucleotide that specifically hybridizes to a complementary polynucleotide sequence.
As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms “polypeptide”, “peptide” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
As used herein, the term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thereupon, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and represent one species of conservatively modified variation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible “silent variation” of the nucleic acid. It is known by persons skilled in the art that each codon in a nucleic acid (except AUG, which is the only codon for the amino acid, methionine; and UGG, which is the only codon for the amino acid tryptophan) can be modified to yield a functionally identical molecule. Therefore, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence. In some embodiments, a nucleotide sequence variant encodes a polypeptide having an altered amino acid sequence.
With respect to amino acid sequences, persons skilled in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
“Transcription” as used herein, refers to the enzymatic synthesis of an RNA copy of one strand of DNA (i.e., template) catalyzed by an RNA polymerase (e.g. a DNA-dependent RNA polymerase).
A “target DNA sequence” is a DNA sequence of interest for which detection, characterization or quantification is desired. The actual nucleotide sequence of the target sequence may be known or not known. Target DNAs are typically DNAs for which the CpG methylation status is interrogated. A “target DNA fragment” is a segment of DNA containing the target DNA sequence. Target DNA fragments can be produced by any method including e.g., shearing or sonication, but most typically are generated by digestion with one or more restriction endonucleases.
The methylated target DNA fragment is typically generated from a sample containing genomic DNA by restriction enzyme digestion. Methods for preparing and digesting genomic DNA with restriction enzymes are well known in the art. Samples suitable for analysis according to the methods of the invention include but are not limited to biological, clinical and biopsy specimens, such as blood, sputum, saliva, urine, semen, stool, bodily discharges, exudates, or aspirates and tissue samples, such as biopsy samples.
The terms “complementary” or “complementarity” are used in reference to a first polynucleotide (which may be an oligonucleotide) which is in “antiparallel association” with a second polynucleotide (which also may be an oligonucleotide). As used herein, the term “antiparallel association” refers to the alignment of two polynucleotides such that individual nucleotides or bases of the two associated polynucleotides are paired substantially in accordance with Watson-Crick base-pairing rules. Complementarity may be “partial,” in which only some of the polynucleotides' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the polynucleotides. Those skilled in the art of nucleic acid technology can determine duplex stability empirically by considering a number of variables, including, for example, the length of the first polynucleotide, which may be an oligonucleotide, the base composition and sequence of the first polynucleotide, and the ionic strength and incidence of mismatched base pairs.
As used herein, the term “hybridization” is used in reference to the base-pairing of complementary nucleic acids, including polynucleotides and oligonucleotides containing 6 or more nucleotides. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, the stringency of the reaction conditions involved, the melting temperature (Tm) of the formed hybrid, and the G:C ratio within the duplex nucleic acid. Generally, “hybridization” methods involve annealing a complementary polynucleotide to a target nucleic acid (i.e., the sequence to be detected either by direct or indirect means). The ability of two polynucleotides and/or oligonucleotides containing complementary sequences to locate each other and anneal to one another through base pairing interactions is a well-recognized phenomenon.
As used herein, “MBP” means methyl binding protein. There are various methyl binding proteins that may be used in accordance with various embodiments described herein, and include but are not limited to, MBD1, MBD2, MBD4, MeCP272 and the Kaison protein family.
As used herein, “MBD” means methyl-CpG-binding domain.
As used herein, the term “promoter” refers to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter can optionally include distal enhancers or repressor elements which can be located several thousand base pairs from the start site of transcription.
As used herein, the term “constitutive promoter” refers to a promoter which is active under most environmental conditions.
As used herein, the term “inducible promoter” refers to a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light.
As used herein, the term “operably linked” includes reference to a functional linkage between a promoter and a nucleic acid sequence, wherein the promoter sequence initiates and/or mediates transcription of the nucleic acid sequence. Generally, operably linked means that the polynucleotide sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
As used herein, the term “recombinant” includes reference to a cell, or nucleic acid, or vector, that has been modified by the introduction of a heterologous nucleic acid or the alteration of a native nucleic acid to a form not native to that cell, or that the cell is derived from a cell so modified. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
As used herein, the term “recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a target cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes a nucleic acid to be transcribed, and a promoter.
As used herein, the term, “specifically binds” includes reference to the preferential association of a ligand, in whole or part, with a particular target molecule (i.e., “binding partner” or “binding moiety” relative to compositions lacking that target molecule). It is, of course, recognized that a certain degree of non-specific interaction may occur between a ligand and a non-target molecule. Nevertheless, specific binding, may be distinguished as mediated through specific recognition of the target molecule. Typically, specific binding results in a much stronger association between the ligand and the target molecule than between the ligand and non-target molecule.
By “fusion protein”, “fusion polypeptide” or “fusion peptide” it is meant a protein composed of a plurality of protein components that while typically unjoined in their native state, are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. “Protein” in this context includes proteins, polypeptides and peptides. Plurality in this context means at least two. It will be appreciated that the protein components can be joined directly or joined through a peptide linker/spacer as known to one skilled in the art. In addition, as outlined below, additional components such as fusion partners including targeting sequences, etc. may be used.
By “reporter protein” or “reporter tag” it is meant a protein that by its presence in or on a cell or when secreted in the media allow the cell to be distinguished from a cell that does not contain the reporter protein. Reporter genes fall into several classes, as outlined above, including, but not limited to, detection genes, indirectly detectable genes, and survival genes.
In a preferred embodiment, the reporter protein is a detectable protein. A “detectable protein” or “detection protein” (encoded by a detectable or detection gene) is a protein that can be used as a direct label; that is, the protein is detectable (and preferably, a cell comprising the detectable protein is detectable) without further manipulations or constructs. As outlined herein, preferred embodiments of screening utilize cell sorting (for example via FACS) to detect reporter (and thus peptide library) expression. Thus, in this embodiment, the protein product of the reporter gene itself can serve to distinguish cells that are expressing the detectable gene. In this embodiment, suitable detectable genes include those encoding autofluorescent proteins.
As is known in the art, there are a variety of autofluorescent proteins known; these generally are based on the green fluorescent protein (GFP) from Aequorea and variants thereof; including, but not limited to, GFP, (Chalfie et al., “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263(5148):802-805 (1994)); enhanced GFP (EGFP; Clontech—Genbank Accession Number U55762)), blue fluorescent protein (BFP; Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; Stauber, R. H., Biotechniques 24(3):462-471 (1998); Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; Clontech Laboratories, Inc., Palo Alto, Calif.) and red fluorescent protein. In addition, there are recent reports of autofluorescent proteins from Renilla and Ptilosarcus species. See WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat. Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558; all of which are expressly incorporated herein by reference.
As used herein, the term “sample” refers to any biological sample obtained from a subject or an individual, cell line, tissue culture, or other source containing polynucleotides or polypeptides or portions thereof. As indicated, biological samples include body fluids (such as blood, sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the polynucleotides of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. A biological sample which includes genomic DNA, mRNA or proteins is preferred as a source.
The present invention provides a variant human methyl binding domain 2 (hMBD2) nucleic acid and amino acid sequence variants and the use of these variants as a simple and sensitive technology for the detection of CpG methylation in DNA. This hMBD2 variants of the invention bind methylated DNA. In particular, the hMBD2 variants of the invention recognize and/or bind DNA comprising a single methylated CpG site, with high affinity.
In one embodiment, the present invention provides isolated nucleic acids of DNA, RNA, and analogs and/or chimeras thereof, comprising a polynucleotide, wherein said polynucleotide encodes a variant human methyl binding domain 2 (hMBD2) polypeptide.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising a sequence selected from:
In one embodiment, the invention provides a polynucleotide which encodes a variant hMBD2 polypeptide of the invention comprising a sequence selected from:
All nucleotide sequences are 5′ to 3′ unless otherwise noted.
The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO; 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO; 35; or SEQ ID NO: 36. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; or SEQ ID NO: 26.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO; 14; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; or SEQ ID NO: 26. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; or SEQ ID NO: 26. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKKEVVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVD LSSFDYRTGKM (SEQ ID NO; 7).
In one embodiment, the invention provides a polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO; 7. The present invention further provides conservatively modified variants of the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 7. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 7.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 7. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 7. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVDL SSFDFRTGKM (SEQ ID NO: 8).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 8 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAATTTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTTTCGTACCGGCAAAATG-3′ (SEQ ID NO: 27). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 27. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 8.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 8. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 8. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVDL SSFDYTGKM (SEQ ID NO: 9).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 9 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAATTTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTATCGTACCGGCAAAATG-3′ (SEQ ID NO: 28). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 28. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 9.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 9. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 9. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKKEEVIRKSGLSAGKIDVYYFSPSGKIRSKPQLARYLGNTVDL SSFDFRTGKM (SEQ ID NO: 10).
In one embodiment, the invention provides the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 10. The present invention further provides conservatively modified variants of the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 10. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 10.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 10. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 10. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKRDVYYFSPSGKKFRSKPQLARYLGNTVD LSSFDFRTGKM (SEQ ID NO: 11).
In one embodiment, the invention provides the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 11. The present invention further provides conservatively modified variants of the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 11. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 11.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 11. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 11. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKIDVYYFSPSGKKFRSKPQLARYLGNTVDL SSFDFRTGKM (SEQ ID NO: 12).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 12 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAATCGATGTGTATTATTTTAGCC CGAGCGGCAAAAAATTTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACACC GTGGATCTGAGCAGCTTTGATTTTCGTACCGGCAAAATG-3′ (SEQ ID NO: 29). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 29. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 12.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 12. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 12. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRTDCPALPPGWKREEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVDL SSFDFRTGKM (SEQ ID NO: 13).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 13 comprises the sequence 5′-GAAAGCGGCAAACGCACGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAATTTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTTTCGTACCGGCAAAATG-3′ (SEQ ID NO: 30). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 30. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 13.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 13. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 13. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGRSAGKIDVYYFSPSGKKIRSKPQLARYLGNTVDL SSFDYRTGKM (SEQ ID NO: 14).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 14 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCGGAGCGCGGGCAAAATCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAAATTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTATCGTACCGGCAAAATG-3′ (SEQ ID NO: 1). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 1. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 14.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 14. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 14. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKSDVYYFSPSGKKFRSKRQLARYLGNTVD LSSFDYRTGKM (SEQ ID NO: 15).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 15 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAATTTCGTAGCAAACGGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTATCGTACCGGCAAAATG-3′ (SEQ ID NO: 31). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 31. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 15.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 15. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 15. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKIDVYYFSPSGKKIRSKPQLARYLGNTVDL SSFDFRTCKM (SEQ ID NO: 22).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 22 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAATCGATGTGTATTATTTAGCC CGAGCGGCAAAAAAATTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACACC GTGGATCTGAGCAGCTTTGATTTTCGTACCTGCAAAATG-3′ (SEQ ID NO; 32). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 32. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 22.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 22. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 22. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKSDVYYFSPSGKKIRSKPQLARYLGNSVDL SSFDYRTGKM (SEQ ID NO: 23).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 23 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAAATTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACTC CGTGGATCTGAGCAGCTTTGATTATCGTACCGGCAAAATG-3′ (SEQ ID NO: 33). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 33. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 23.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 23. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 23. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKSDVYYYSPSGKKFRSKPQLARYLGNTVD LSSFDYRTGKM (SEQ ID NO: 24).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 24 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTATAGC CCGAGCGGCAAAAAATTTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTATCGTACCGGCAAAATG-3′ (SEQ ID NO: 34). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 34. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 24.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 24. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 24. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In a one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKSDVYYFSPSGKKIRSKPQLARYLGNTVDL SSFDYRTGKM (SEQ ID NO: 25).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 25 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATCCGTAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAAATTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTATCGTACCGGCAAAATG-3′ (SEQ ID NO: 35). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 35. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 25.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 25. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 25. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In one embodiment, the invention provides a variant hMBD2 polypeptide comprising the sequence ESGKRMDCPALPPGWKREEVIRKSGLSAGKIDVYYFSPSGKKIRSKPQLARYLGNTVDL SSFDYRTGKM (SEQ ID NO: 26).
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 26 comprises the sequence 5′-GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCTGAGCGCGGGCAAAATCGATGTGTATTAITTTTAGCC CGAGCGGCAAAAAAATTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACACC GTGGATCTGAGCAGCTTTGATTATCGTACCGGCAAAATG-3′ (SEQ ID NO: 36). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 36. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 26.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 26. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 26. In one embodiment, such a modified polypeptide binds a DNA sequence having a single methylated CpG site with a dissociation constant (Kd) of greater than or equal to 3.1±1.0 nM. The dissociation constant can be determined by one skilled in the art using routine methods, for example, as those described herein.
In one embodiment, the polynucleotide which encodes the variant hMBD2 polypeptide of SEQ ID NO: 14 comprises the sequence (GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGTTGGAAAAGAGAAG AAGTGATTCGTAAAAGCGGCCGGAGCGCGGGCAAAATCGATGTGTATTATTTTAGC CCGAGCGGCAAAAAAATTCGTAGCAAACCGCAGCTGGCGCGTTATCTGGGCAACAC CGTGGATCTGAGCAGCTTTGATTATCGTACCGGCAAAATG/SEQ ID NO: 1). The present invention further provides conservatively modified variants of the polynucleotide of SEQ ID NO: 1. It is known in the art that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid sequence. These “silent variations” can be used to encode the polypeptide of SEQ ID NO: 14.
In one embodiment, the present invention provides a variant hMBD2 polypeptide of SEQ ID NO: 14. In another embodiment, the present invention provides a conservatively modified variant of the polypeptide of SEQ ID NO: 14 provided that such a modified polypeptide binds a DNA sequence having a single methylated CpG site with at a binding affinity (Kd) of at least 3.1±1.0 nM. The dissociation constant/binding affinity can be determined by one skilled in the art using routine methods, for example, as those described herein.
The present invention further provides fusion proteins that bind to methylated CpG DNA. Such fusion proteins comprise a variant hMBD2 polypeptide of the invention and a reporter protein. In one embodiment, the variant hMBD2 polypeptide comprises a sequence selected from SEQ ID NO: 7; SEQ ID NO: 8. SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 22; SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 25; or SEQ ID NO: 26.
The present invention further provides fusion proteins that bind to methylated CpG DNA. Such fusion proteins comprise a variant hMBD2 polypeptide of the invention and a reporter protein. In one embodiment, the variant hMBD2 polypeptide comprises SEQ ID NO: 14.
The present invention further provides fusion proteins that bind to methylated CpG DNA. Such fusion proteins comprise a variant hMBD2 polypeptide of the invention and a reporter protein. In one embodiment, the variant hMBD2 polypeptide comprises SEQ ID NO: 23.
Also provided are polynucleotides encoding the fusion polypeptides of the invention. In some embodiments, the nucleic acid molecule of the present invention is part of a vector. The present invention relates in another embodiment to a vector comprising the nucleic acid molecule of this invention. Such a vector may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker or reporter genes which allow for the selection and/or replication and/or detection of said vector in a suitable host cell and under suitable conditions. In one embodiment, said vector is an expression vector, in which the nucleic acid molecule of the present invention is operatively linked to an expression control sequence(s) (e.g., a promotor) allowing expression in prokaryotic or eukaryotic host cells as described herein.
These variant hMBD2 sequences can be incorporated into vectors as multimerized constructs with a reporter (e.g., an enhanced green fluorescent protein (eGFP)) tag. For example, single peptides with 2-500, preferably 2-250, preferably 2-100, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 copies of the variant hMBD2 polypeptides according to the invention and a C-terminal reporter (e.g., eGFP) tag can be prepared. In one embodiment, the variant hMBD2 polypeptide comprises SEQ ID NO: 14. In one embodiment, the variant hMBD2 polypeptide comprises SEQ ID NO: 23.
The present invention relates also to an in vitro method for detecting methylated DNA comprising contacting a sample comprising methylated and/or unmethylated DNA with the polypeptide of the present invention; and detecting the binding of said polypeptide to methylated DNA.
In one embodiment, said in vitro method is reverse South-Western blotting, immune precipitation, affinity purification of methylated DNA or Methyl-CpG-immunoprecipitation (MCIp). However, said in vitro method is not limited thereto, but could basically be any procedure in which the polypeptide of the present invention is linked to a solid matrix, for example, a matrix such as sepharose, agarose, capillaries, vessel walls, as is also described herein in connection with the diagnostic composition of the present invention.
In another embodiment, the aforementioned in vitro methods further comprise as step (c) analyzing the methylated DNA, for example, by sequencing, Southern Blot, restriction enzyme digestion, bisulfite sequencing, pyrosequencing or PCR. Yet, analyzing methylated DNA which has been isolated, enriched, purified and/or detected by using the polypeptide of the present invention is not limited to the aforementioned methods, but encompasses all methods known in the art for analyzing methylated DNA, e.g., RDA, microarrays and the like.
In some embodiments, detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc. The use of a fluorescent tag (e.g., eGFP and HA tags) allow the variant hMBD2 proteins of the invention to transduce binding to methylated DNA to a directly observable signal which reduces assay complexity, reduces time, and eliminates the need for DNA sequencing.
Accordingly, in one embodiment the composition according to the invention is a diagnostic composition, optionally further comprising suitable means for detection.
A further embodiment of the present invention is the use of the polypeptide of the present invention for the detection of methylated DNA.
In addition, the nucleic acid molecules, the polypeptide, or the vector, of the present invention are used for the preparation of a diagnostic composition for detecting methylated DNA.
Additionally, the present invention provides a kit comprising the nucleic acid molecule, the vector, or the polypeptide of the present invention.
Advantageously, the kit of the present invention further comprises, optionally (a) reaction buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of scientific or diagnostic assays or the like. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
The kit of the present invention may be advantageously used, inter alia, for carrying out the method for isolating, enriching, purifying and/or detecting methylated DNA as described herein and/or it could be employed in a variety of applications referred herein, e.g., as diagnostic kits, as research tools or therapeutic tools. Additionally, the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to detect DNA methylation.
The cDNA encoding the hMBD2 gene (AAs 145-213/SEQ ID NO: 16) was PCR amplified from the pMal-c2X-MBD2 construct (Porter et al., 2007) from Indraneel Ghosh (University of Arizona). The forward 5′-TAC AGC TAG CGA AAG CGG CAA ACG-3′ (SEQ ID NO: 17), and reverse 5′-GAC AGG ATC CCA TTT TGC CGG TAC GA-3′ (SEQ ID NO: 18) primer pair was designed to append flanking 5′ NheI and 3′ BamHI restriction sites. The PCR reaction was carried out as described above. The thermocycling profile was as follows: initial denaturation at 98° C. for 30 sec followed by 30 cycles of denaturation at 98° C. for 10 sec, annealing at 60° C. for 30 sec, extension at 72° C. for 30 sec, and a final extension at 72° C. for 10 min. All other steps were performed as described above.
To establish a platform for characterizing and engineering methyl binding domain family proteins, cDNA encoding the MBD domain from hMBD2 was cloned into the pCTCON-2 yeast surface display vector. The construct is expressed as a fusion consisting of Aga2p (for yeast cell surface attachment), HA, MBD, and c-Myc (Chao, Lau, Hackel, Sazinsky, Lippow and Wittrup, 2006) (
The hMBD2 protein was screened across a range of methylated DNA concentrations to assess relative binding affinities (data not shown). Subsequently, equilibrium binding titration was used to quantitatively determine the affinity and selectivity of the methyl-CpG binding domain of hMBD2. In addition to an anti-c-Myc/ALEXA FLUOR® 488 antibody pair used to show surface display expression, yeast was equilibrated with biotinylated DNA at various concentrations followed by secondary labelling with streptavidin, ALEXA FLUOR® 647 (
Quantitative equilibrium binding of DNA to yeast displayed hMBD2 proteins was determined using the method described previously (Chao et al., 2006). EBY100 transformed with pCTCON-2/hMBD2 were grown in SDCAA media overnight at 30° C. and 250 rpm. After reaching OD600=2-5, cultures were inoculated to OD600=1 in SGCAA and incubated at 20° C. and 250 rpm for 40-48 h to induce surface display fusion expression. Induced EBY100 were resuspended to OD600=1 in PBSA (1×PBS, 0.1% w/v BSA). Five-hundred thousand EBY100 cells in PBSA were incubated with pre-hybridized DNA (synthesized by Integrated DNA Technologies) at concentrations ranging from 0.06-100 nM in volumes of PBSA ranging from 2225-200 μL to provide a 10-fold molar excess of DNA relative to the number of surface display fusions assuming 5×104 MBD/cell (Chao, Lau, Hackel, Sazinsky, Lippow and Wittrup, 2006). The DNA oligonucleotides used for characterizing the variant hMBD2 polypeptides were derived from the MGMT gene as described previously (Yu, Blair, Gillespie, Jensen, Myszka, Badran, Ghosh and Chagovetz, 2010) and functionalized with biotin on the 5′ end of each target strand to facilitate fluorescence labelling (
Equilibrium binding was performed at room temperature for 45 min as described previously (Chao, Lau, Hackel, Sazinsky, Lippow and Wittrup, 2006). The binding of methylated DNA to displayed hMBD2 proteins was detected using streptavidin, ALEXA FLUOR® 647 (Life Technologies), and the fraction of EBY100 that expressed the surface display fusions was identified using the chicken anti-cMyc (Gallus Immunotech)/ALEXA FLUOR® 488 goat anti-chicken (Life Technologies) antibody pair. The dissociation constant (Kd) for each oligonucleotide was determined from an equilibrium binding titration curve fit obtained after plotting the mean fluorescence of the EBY100 cells displaying MBDs versus each DNA concentration (Chao, Lau, Hackel, Sazinsky, Lippow and Wittrup, 2006). Each reported Kd value is the average of three biological replicates performed on separate days following the same protocol.
The equilibrium dissociation constant for each oligo was determined by fitting the normalized mean fluorescence versus DNA concentration data for each of three biological replicates (
The GeneMorph II Random Mutagenesis Kit (Agilent) was used to perform epPCR on the hMBD2 gene. To affect 1-3 mutations per MBD2 gene (˜5-15 mutations/kb), 250 ng of target DNA (7.75 μg plasmid construct) was used as the template for the epPCR reaction. The forward 5′-CGA CGA TTG AAG GTA GAT ACC CAT ACG ACG TTC CAG ACT ACG CTC TGC AG-3′ (SEQ ID NO: 19), and reverse 5′-CAG ATC TCG AGC TAT TAC AAG TCC TCT TCA GAA ATA AGC TTT TGT TC-3′ (SEQ ID NO: 20) primer pair (Chao, Lau, Hackel, Sazinsky, Lippow and Wittrup, 2006) was used to produce a 367 bp product. The PCR reaction contained 1×Mutazyme II reaction buffer (Agilent), 40 nmol of each dNTP (New England BioLabs), 125 ng of each primer (Integrated DNA Technologies), 7.75 μg pCTCON-2/hMBD2 construct, and 2.5 U Mutazyme II DNA polymerase (Agilent) in a final volume of 50 μL. The thermocycling profile was as follows: initial denaturation at 95° C. for 2 min followed by 30 cycles of denaturation at 95° C. for 30 sec, annealing at 58° C. for 30 sec, extension at 72° C. for 1 min, and a final extension at 72° C. for 10 min. The epPCR product was gel purified and amplified using standard Taq based PCR to provide sufficient DNA material for library creation via transformation and homologous recombination in EBY100 yeast cells (Chao, Lau, Hackel, Sazinsky, Lippow and Wittrup, 2006).
A random mutant yeast display library of 108 hMBD2-derived clones was created and screened to isolate novel MBD proteins exhibiting increased binding affinity to DNA containing at least one methylated CpG dinucleotide.
The library was screened by incubating a number of EBY100 cells 10-fold greater than the calculated diversity (Chao, Lau, Hackel, Sazinsky, Lippow and Wittrup, 2006). For the first library this corresponded to 2×109 cells for a diversity of 2×108. After the first round of fluorescence activated cell sorting (FACS), the number of cells screened was 10-fold greater than the number collected from the previous sort. Because the starting hMBD2 Kd was less than 10 nM, the library was enriched for high affinity MBD2 variants using a kinetic screen (Boder and Wittrup, 1998). The library was incubated with 100 nM biotinylated omo dsDNA while ensuring a 10-fold molar excess of DNA for 45 min at room temperature in order to saturate surface displayed MBDs with labeled DNA. The cells were then washed, resuspended in PBSA, and incubated with 100 nM unlabeled, competitor omo dsDNA at room temperature to distinguish clones by differences in the degree of labeling due to varying dissociation rate constants and, therefore, binding affinities; concurrently, the cMyc epitope tag of each surface display fusion was labeled with chicken anti-cMyc IgY diluted 1:250. The competition time was determined using the method described previously (Boder and Wittrup, 1998) and increased in successive rounds in the range of 90-120 min. The EBY100 population was washed and labeled using streptavidin, ALEXA FLUOR® 647 and ALEXA FLUOR® 488 goat anti-chicken secondary reagents (both diluted 1:100) on ice for 15 min. The library was washed and resuspended to a density of 107 cells/mL in sterile PBSF for sorting on a MoFlo XDP (Beckman Coulter). Diagonal sort gates were drawn to specify the fraction of the cells collected. This value was decreased from 5%, to 1%, and to 0.1-0.2% over three consecutive rounds of flow cytometry following the method described previously (Boder and Wittrup, 1998). Yeast cells were collected in SDCAA media and subsequently propagated at 30° C. and 250 rpm. A tenfold oversampling of the expanded cells was resuspended in SGCAA media for surface display fusion expression and sorting in the next round of screening. After the third round of FACS, the plasmids encoding the MBD2-derived variants were collected using the ZYMOPREP™ Yeast Plasmid Miniprep II (Zymo Research) and transformed into Mach 1 E. coli cells (Life Technologies). Individual clones were isolated and the MBD2 gene was sequenced using the forward primer 5′-CCC CTC AAC TAG CAA AGG CAG-3′ (SEQ ID NO: 21).
The library was screened by DNA dissociation kinetics such that clones with reduced off rates retained more biotinylated DNA, exhibited greater fluorescence when fluorescently labeled, and were separated using FACS (Boder and Wittrup, 1998). After the first round of epPCR, individual clones were isolated and the gene encoding each MBD variant was sequenced. Six amino acid substitutions (Table II) which combined to produce five unique MBD variants having one or two mutations each (
The sequence of the MBD variant ¼, having the highest observed binding affinity, was aligned with the wild-type primary structure (
After screening the first library, the plasmids collected from the final sort were subjected to a second round of mutagenesis by epPCR as described above to create another library with a calculated diversity of 1×108. This second library was screened using the same protocol above for the purpose of finding additional mutations giving rise to higher affinity MBD proteins.
Three new amino acid substitutions were observed following this round of evolution (Table II) as well as new combinations of mutations observed previously. The K161R mutation was present in every variant sequenced, and the F208Y was found in 67% of variants up from a 20% frequency in the first round. The four new MBD variants had two to five mutations each (
The cDNA for MBD2 variant 2/5 was codon optimized for expression in E. coli (Gene Art-Life Technologies) and used to create an MBD-GFP fusion analogous to that reported previously (Yu, Blair, Gillespie, Jensen, Myszka, Badran, Ghosh and Chagovetz, 2010). The protein consists of an N-terminal His6-tag followed by the nuclear localization sequence PKKKRKV, the MBD2 variant 2/5, a hemagglutinin (HA) tag, and a C-terminal enhanced green fluorescent protein (GFP) tag. A BsaI restriction site was included immediately preceding the MBD2 variant 2/5 to facilitate concatenation. The cDNA encoding the fusion was synthesized as a gBlock with flanking 5′ EcoRI and 3′ XhoI restriction sites plus four nucleotide overhangs, double digested, ligated into the pET-30b+ vector, and transformed into Mach 1 E. coli cells (Life Technologies). The miniprepped plasmid was subsequently transformed into BL21 (DE3) Tuner E. coli cells (Novagen) for expression.
To create the MBD2 variant 2/5 multimer, a second gBlock consisting of the codon optimized cDNA for the MBD followed by the cDNA for a (Gly4-Ser)2 linker with flanking 5′ and 3′ BsaI restriction sites plus six nucleotide overhangs on each end was designed. Both the pET-30b+/MBD2 variant 2/5 plasmid and second gBlock were digested with BsaI (New England Biolabs) and ligated using T4 DNA ligase (New England Biolabs) such that the digested gBlock was in large molar excess. The ligation product was transformed into Mach 1 E. coli cells and plated onto LB agar plates supplemented with kanamycin. Individual clones were screened for the number of incorporated MBD variant 2/5 monomer units on the basis of the size of the fragment obtained following double digestion with EcoRI and XhoI. The plasmid encoding the 3×MBD2 variant 2/5-GFP protein was transformed into BL21 (DE3) Tuner E. coli cells (Novagen) for expression. The 1× and 3×MBD2 variant 2/5 proteins were expressed (Boyd et al., 2012) and purified under denaturing conditions with on-column refolding (Jorgensen, Adie, Chaubert and Bird, 2006) using the protocols described previously.
Clear glass slides coated with an agarose film were prepared (Afanassiev et al., 2000) and printed (Heimer, Shatova, Lee, Kaastrup and Sikes, 2014) with pre-hybridized ooo probe/ooo target, omo probe/omo target, and omm probe/omm target oligonucleotides at 10 μM concentration in 3×SSC as described previously. A circular, 9 mm diameter isolator well was cut from Scotch 3M 665 tape and affixed to the biochip to define each test area. Each biochip was then rinsed under a stream of DI water and blown dry using compressed nitrogen gas. Biochips ready for testing were stored in the vacuum desiccator until needed.
N×MBD proteins were diluted in binding buffer (20 mM HEPES, pH 7.9, 3 mM MgCl2, 10% v/v glycerol, 1 mM dithiothreitol, 100 mM KCl, 0.1% w/v BSA, 0.01% Tween-20, and 1 μM ssDNA) and pre-incubated for 10 min at room temperature. Each 40 μL N×MBD dilution was added to a separate test area and incubated for 40-45 min in a humid chamber at ambient temperature (approximately 20-22° C.). Each slide was washed sequentially with 1×PBS/0.1% v/v Tween 20 (PBST), 1×PBS, and 18 M. DI water and blown dry using compressed nitrogen gas. The monoclonal mouse HA.11 clone 16B12 antibody (BioLegend) was diluted 1:100 in 1×PBS/0.1% w/v BSA (PBSA), added to each test area, and incubated for 10 min at 4° C. in a humid chamber pre equilibrated to temperature. The slide was washed and dried as described previously. The secondary ALEXA FLUOR® 647 goat, anti-mouse antibody was diluted 1:100 in PBSA, added to each test area, and incubated for 10 min at 4° C. in a humid chamber pre equilibrated to temperature. The slide was washed and dried as described previously before scanning with a GenePix 4000B fluorescent microarray scanner (Molecular Devices). Each fluorescence image was analyzed using ImageJ (NIH). The mean fluorescence intensity for each spot was determined by adjusting the threshold of the image to include the entire spot area and averaging the constituent pixel intensities. The values for all spots of the same DNA methylation pattern were averaged and plotted versus the N×MBD concentration in order to fit the data and determine the apparent equilibrium dissociation constant Kd,app.
In order to determine the molecular basis of the observed affinity improvements, the SWISS-MODEL system (Biasini et al., 2014) and the published chicken MBD2 NMR structure (2KY8 PDB) (Scarsdale, Webb, Ginder and Williams, 2011) was used to generate a homology model of the MBD2 variant 2/5. The kinetic library screening method is used to isolate variants with decreased off-rates (Boder and Wittrup, 1998). As such, forming new, non-covalent protein-DNA interactions slows the rate of MBD-DNA dissociation and results in improved binding affinity. In the case of hMBD2, mutation of phenylalanine to tyrosine at the 208 position adds a para substituted hydroxyl group to the aromatic side chain which donates a hydrogen bond to the DNA phosphate backbone (
The frequency of which the K161R mutation is observed, if used as a surrogate for fitness, may indicate it is the most significant residue of those found affecting MBD binding affinity. Despite being the highest affinity wild-type MBD reported (Fraga, Ballestar, Montoya, Taysavang, Wade and Esteller, 2003), MBD2 is the only wild-type human or mouse MBD having a lysine at this position instead of an arginine. The hMBD2 K161 side chain forms a single hydrogen bond between its e-amino group and the backbone of G211 in the wild-type protein (Scarsdale, Webb, Ginder and Williams, 2011). Mutating this residue to arginine substitutes a resonance stabilized guanidium group for the e-amino which allows for the formation of a second hydrogen bond to the backbone of D151 (
The two mutations to isoleucine S1751 and F187I appear to exist within a similar context in the MBD structure. Both are adjacent to residues known to form base-specific interactions: K174 with the guanine downstream of the CpG and R188 directly with the methylated CpG, respectively (Scarsdale, Webb, Ginder and Williams, 2011). D176 was also shown to form a CH . . . O hydrogen bond to the methyl group of 5 mC in homologous h/mMeCP2 over a similar distance (˜3.5 Å) (Ho, McNae, Schmiedeberg, Klose, Bird and Walkinshaw, 2008). Further, I187 is one member of the four amino acid sequence KIRS in which all three other residues interact with the bound DNA strand. In both instances, the hydrophobic isoleucine side chains are oriented nearly opposite of those interacting with the DNA. The I175 side chain appears to engage in a hydrophobic interaction with I165 at the C-terminal end of the second β-strand (
Starting with a wild-type mMBD1 (Kd=30 μM), others have reported a 60-fold improvement in MBD affinity (Kd=0.5 μM) for singly methylated DNA by concatenating four mMBD1s into a single peptide (Jorgensen, Adie, Chaubert and Bird, 2006). Adopting this established method, the highest affinity monomeric MBD variant (MBD 2/5,
In order to further the development of high-performance, interfacial epigenotyping assays (Heimer, Shatova, Lee, Kaastrup and Sikes, 2014, Yu, Blair, Gillespie, Jensen, Myszka, Badran, Ghosh and Chagovetz, 2010), N×MBD (i.e., multimeric) variants were evaluated on agarose coated slides (Afanassiev, Hanemann and Wölfl, 2000) with immobilized dsDNA having no (ooo), one (omo), or two (omm) methylated CpG dinucleotides. The bound MBDs were labeled with an anti-HA/ALEXA FLUOR® 647 antibody pair and scanned them (
Such binding affinity improvements while maintaining specificity allows us to preserve solution-like binding characteristics in a useful interfacial format where surface effects as well as MBD loss during wash steps can reduce the fractional MBD coverage. The fractional coverage of single methylated CpGs as a function of concentration for MBD proteins with varying Kd was estimated using a Langmuir adsorption model (
Developing a protein that will recognize hemi-methylated DNA, where the cytosine bases are only methylated on one of the two DNA strands, would allow the detection of a methylated sequence from a patient's sample bound to an unmethylated capture probe. A library created from human MBD2 as described above was used as a starting point. Variants with improved affinity for hemi-methylated DNA were isolated and analyzed in a yeast surface display construct.
Characterization of Binding Affinities Using Yeast Surface Display MBD proteins were displayed on the surface of EBY100 S. cerevisiae yeast cells as described above. The cells containing the pCTCON-2 vector with the MBD insert were grown overnight in SDCAA medium at 30° C. and 250 rpm. To induce protein expression, after the SDCAA cultures reached an OD600 between 2 and 5, the cells were resuspended in SGCAA medium to an OD600 of 1 and incubated at 20° C. and 250 rpm for 36-48 hours. The cells were then resuspended in PBS with 0.1% BSA and an equilibrium binding titration was performed by incubating the cells expressing the MBD protein with biotinylated DNA oligomers at a range of concentrations between 0.05 and 100 nM for 45 min at room temperature. Total reaction volumes were chosen to ensure 10-fold excess of DNA in each sample, calculated based on the protein expression level identified by Chao et al. (Chao et al., 2006). Expressed protein and bound DNA were labeled with chicken anti-cMyc/AlexaFluor-488 and streptavidin-AlexaFluor-647, respectively. The extent of binding was evaluated using flow cytometry, and dissociation constants were calculated using the method described by Chao et al. (Chao et al., 2006) Screening the MBD Library for Improved Affinity for Hemi-Methylated DNA To enrich for protein variants that bind to hemi-methylated DNA, biotinylated DNA with a single methylated cytosine on one strand was incubated with streptavidin conjugated magnetic beads. The DNA concentration in the 1 ml reaction was 55 nM. A total of 4×109 cells expressing the MBD library were incubated with the DNA covered beads for 2 hours at 4° C. to capture those expressing proteins with good binding characteristics. After the incubation, the beads with cells attached were separated from unbound cells with a magnet and resuspended in SDCAA medium, pH 4.5, supplemented with pen-strep (1:100 dilution). The captured cells were grown overnight at 30° C. and 250 rpm. The bead selection was repeated with 2×10s cells from the enriched library. After the second selection with magnetic beads, the cells were again grown up and protein expression was induced. Two additional selections for hemi-methylated DNA were performed using fluorescence-activated cell sorting (FACS). For the first FACS selection, binding reactions were prepared as described for characterization by flow cytometry and a gate was drawn during sorting to capture the top 1% of cells. This top 1% was defined using a diagonal sort window, as described by Chao et al. (Chao et al., 2006). In the second FACS selection, the top 0.37% of the cells were isolated. The plasmids encoding the selected proteins were extracted using the ZYMOPREP™ Yeast Plasmid Miniprep II kit, transformed into Mach 1 E. coli, and grown on LB plates containing 100 μg/ml ampicillin. Ten single colonies were selected, and for each of these colonies, the MBD insert was sequenced. After sequencing, plasmids containing unique clones were transformed into EBY100 S. cerevisiae and expressed on the surface. To compare the clones, binding reactions were performed as described above with two DNA concentrations, 10 nM and 50 nM. After a comparison of binding affinities among the isolated clones, titrations were performed to determine the dissociation constant of the top performing variant.
The sequence encoding the top performing variant, h4 (see Table 3 below), was PCR amplified from the pCTCON-2 vector using Phusion HF polymerase with the forward primer 5′-GCCTGAATTCTGAAAGCGGCAAACG-3′, which includes an EcoRI restriction site, and the reverse primer 5′-CATTTTGCCGGTACGATAATCAAAGCTGCTC-3′. In this reaction, the DNA was denatured at 95° C. for 6 min, then 30 cycles were performed with 30 sec each of denaturation at 95° C., annealing at 56° C., and extension at 72° C. A 10 min final extension was performed at 72° C. Splicing by overlap extension was used to append an eGFP tag and a biotin accepter sequence to MBD2 variant h4. First, a 3-primer PCR reaction was used to add a linker sequence to the MBD variant. This reaction used the forward primer 5′-GCCTGAATTCTGAAAGCGGCAAACG-3′, the long reverse primer 5′-CGTAGTCTGGCACGTCGTATGGGTACATTTTGCCGGTACGATAATCAAAGCTG-3′ for adding the linker group, and the short reverse primer 5′-CGTAGTCTGGCACGTCGTATGGG-3′ for amplifying the product containing the linker group with the same PCR conditions as the first reaction. The eGFP tag and biotin accepter sequence were amplified from another plasmid using the forward primer 5′-TACCCATACGACGTGCCA-3′and the reverse primer 5′-TGGTGCTCGAGTTTATTCATGC-3′, which added an XhoI restriction site. The eGFP reaction proceeded as described above except the annealing temperature was reduced to 52° C. and the extension time increased to 1 min. For the splicing by overlap extension reaction, the forward primer 5′-GCCTGAATTCTGAAAGCGGCAAACG-3′ and reverse primer 5′-TGGTGCTCGAGTTTATTCATGC-3 were used to amplify the full MBD-GFP fusion protein using touchdown PCR. An annealing temperature of 61° C. was used for the first cycle, and this temperature was decreased by 1° C. for each of the next eight cycles. The final annealing temperature of 53° C. was then used for an additional 30 cycles. The resulting PCR product was cloned into the pET30b vector using the EcoRI and XhoI restriction sites. The pET30b vector containing the insert was transformed into DE3 Tuner E. coli and grown in LB broth supplemented with Kanamycin. To express the fusion protein, the cells were grown in TB medium to an OD600 of 0.6 and then protein expression was induced by the addition of 0.05 mM IPTG. The cells were incubated at 20° C. for 16 hours, pelleted, and lysed using BugBuster HT protein extraction reagent according to the manufacturer's protocol for soluble protein.
Glass slides were coated with 0.2% SEAKEM® LE agarose (Lonza) and arrays of pre-hybridized DNA were printed, as described by Heimer et al. (Heimer et al., 2014). Each slide contained rows with ooo probe/omm target, omo probe/ooo target, and ooo probe/ooo target DNA. The slides were left to dry in a vacuum desiccator overnight. Wells were cut from Scotch 3M tape and placed around the arrays on the slide. The wells were rinsed with 18 MΩ DI water and dried under compressed air. Blocking was performed by incubating the wells with 40 μl of 1% BSA at room temperature for 15 min. After the blocking reaction, the wells were rinsed with PBS and 18 MΩ DI water and dried with compressed air before 40 ul of the clarified cell lysate containing MBD2 variant h4, diluted in binding buffer (20 mM HEPES, pH 7.9, 3 mM MgCl2, 10% (v/v) glycerol, 1 mM dithiothreitol, 100 mM KCl, 0.1% (w/v) BSA, 0.01% Tween-20), was added. The DNA arrays and protein solution were incubated at room temperature for 45 minutes, after which the wells were washed consecutively with PBS/0.1% Tween 20, PBS, and 18 M DI water and dried with compressed air. Bound protein was labeled with streptavidin-ALEXA-FLUOR® 647 diluted 1:100 in PBSA for 10 min at 4° C. and the wells were washed and dried again, as described above. All incubation steps were performed in a humid chamber that had been equilibrated to the desired incubation temperature. Fluorescence was detected using a GenePix 4000B scanner (Molecular Devices) with 635 nm excitation. Quantitative results were obtained by calculating the mean fluorescence and background fluorescence for each spot within the DNA array using the GenePix 6.1 software. For each methylation pattern, the fluorescence intensity was averaged over all of the spots within the well.
To characterize the binding affinity, the MBD proteins were displayed on the surface of S. cerevisiae using the pCTCON-2 vector. The binding affinity of wild-type human MBD2 toward a DNA oligo with a single methyl group on one strand was evaluated using equilibrium binding titrations with flow cytometry. The sequence and methylation patterns of the test DNA used for characterization are shown in
Beginning with the error-prone PCR library generated as described above, variants of the protein human MBD2 were displayed on the surface of yeast cells, and those with improved affinity for hemi-methylated DNA were selected using an equilibrium binding assay. The selection process is depicted in
The amino acid sequences of the proteins isolated after the selection procedure are shown in Table 3. All of the variants isolated had the K161R mutation and 70% had the F208Y mutation, two mutations that, without wishing to be bound by any particular theory, allow for the formation of an additional hydrogen bond to stabilize the protein structure and to bind to the DNA backbone, respectively. The F187I mutation, which is adjacent to the arginine residue that interacts with the methylated cytosine base, was also found in 50% of the isolated proteins.
Unique protein variants were compared (data not shown), and the top-performing protein, variant h4, was characterized by equilibrium binding titrations.
The fourth mutation, T200S, in variant h4, is a small change from a threonine to the slightly smaller serine and is located far from the DNA binding site. This residue is not conserved across the MBD family: it is found as alanine in MBD1, threonine in human MBD2, asparagine in MBD4, and valine in MeCP2. However, none of the wild type MBD proteins nor any of the proteins isolated from the library except for variant h4 have the S200 residue. Nevertheless, this mutation appears to play an important role in binding to hemi-methylated DNA.
To determine whether the new protein can function to distinguish between hemi-methylated and unmethylated DNA in the interfacial binding assays, binding experiments were performed with soluble MBD2 variant h4 and DNA arrays printed on agarose-coated glass slides. The MBD2 variant h4 was cloned into the pET30b bacterial expression vector and expressed as a fusion protein with eGFP and a biotin acceptor sequence. The slides were printed with hemi-methylated DNA as well as unmethylated DNA. Biotinylated MBD bound to the DNA was labeled with streptavidin, ALEXAFLUOR® 647 and detected by fluorescence imaging. In the resulting image, found in
These results demonstrate that variants of the present invention can be used in place of the wild-type MBD proteins used in previously developed epigenotyping assays and that unmethylated DNA probes can now be used instead of methylated probes in these assays. Because methylated DNA probes must be specially synthesized and are much more costly than unmethylated probes, an assay that doesn't require them can be developed more quickly and easily into a method suitable for clinical use. Such binding assays could be extremely valuable as an alternative to the chemical conversion-based methods currently used for clinical methylation analyses that have many disadvantages, such as DNA degradation during sample treatment.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 62/183,479, filed on Jun. 23, 2015. The entire teachings of the above application are incorporated herein by reference.
This invention was made with Government support under Grant No. P30 ES002109 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62183479 | Jun 2015 | US |
