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EP3010929A1 - Procédés universels de profilage de la méthylation - Google Patents

Procédés universels de profilage de la méthylation

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Publication number
EP3010929A1
EP3010929A1 EP14812900.0A EP14812900A EP3010929A1 EP 3010929 A1 EP3010929 A1 EP 3010929A1 EP 14812900 A EP14812900 A EP 14812900A EP 3010929 A1 EP3010929 A1 EP 3010929A1
Authority
EP
European Patent Office
Prior art keywords
alkyl
methyltransferase
double
aryl
stranded dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14812900.0A
Other languages
German (de)
English (en)
Other versions
EP3010929A4 (fr
Inventor
Timothy H. Bestor
Jingyue Ju
Xiaoxu Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Columbia University in the City of New York
Original Assignee
Columbia University in the City of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Columbia University in the City of New York filed Critical Columbia University in the City of New York
Publication of EP3010929A1 publication Critical patent/EP3010929A1/fr
Publication of EP3010929A4 publication Critical patent/EP3010929A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • the human genome contains -28 million CpG sites, about 70% of which are methylated at the 5 position of the cytosine (Edwards et al . , 2010) .
  • Aberrant DNA methylation has been linked to a growing list of developmental diseases, age-related neurodegenerative disorders, diabetes and cancer (Robertson et al . , 2005).
  • epigenetic changes in DNA methylation status are increasingly being studied for their role in both normal and disease-associated phenotypic changes, including the Roadmap Epigenomics Project being launched by NIH to create reference epigenomes for a variety of cell types.
  • NIH Roadmap Epigenomics Project
  • BGS Bisulfite genomic sequencing
  • the present invention provides a compound having the structure :
  • R 1( R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • the present invention also provides a composition of matter comprising a compound having the structure:
  • R is wherein X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • the present invention also provides a process of producing a derivative of a double-stranded DNA comprising contacting the double-stranded DNA with a CpG methyltransferase and an S- adenosylmethionine analog having the structure:
  • R is a chemical group capable of being transferred from the S-adenosylmethionine analog by the CpG methyltransferase to a 5- carbon of a non-methylated cytosine of the double-stranded DNA, under conditions such that the chemical group covalently binds to the 5-carbon of the non-methylated cytosine of the double-stranded DNA, and thereby produces the derivative of the double-stranded DNA, wherein the chemical group has the structure:
  • R 1# R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • the present invention also provides a method of determining whether a cytosine present within a double-stranded DNA sequence of known sequence is non-methylated comprising:
  • R is a chemical group capable of being transferred from the S-adenosylmethionine analog by the CpG methyltransferase to a 5 carbon of a non-methylated cytosine of the double-stranded DNA so as to covalently bond the chemical group to the 5 carbon of the non-methylated cytosine of the double-stranded DNA, thereby making a derivatized double stranded DNA, wherein the chemical group has the structure :
  • R lf R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 /
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • step b) sequencing the single strand so obtained in step b) ; and comparing the sequence of the single strand determined in step c) to the sequence of a corresponding strand of the double- stranded DNA of which a derivative has not been produced, wherein the presence of a uracil analog in the single strand of the derivative single strand instead of a cytosine at a predefined position in the corresponding strand of the double- stranded DNA of which a derivative has not been produced indicates that the cytosine at that position in the double- stranded DNA is non-methylated.
  • the present invention also provides a derivatized DNA molecule, wherein the derivatized DNA molecule differs from DNA by comprising a nucleotide residue which comprises a base having the following
  • R x , R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ' ;
  • R' ' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • the present invention also provides a derivatized DNA molecule, wherein the derivatized DNA molecule differs from DNA by comprising a nucleotide residue which comprises a base having the following
  • R 17 R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • sugar is a sugar of the nucleotide residue
  • the present invention also provides a kit for derivatizing a double- stranded DNA molecule or for determining whether a cytosine present within a double-stranded DNA sequence of known sequence is non- methylated comprising: a) a compound having the structure:
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • the present invention also provides a method of determining whether a cytosine present within a double-stranded DNA sequence of known sequence is non-methylated comprising:
  • R is a chemical group capable of being transferred from the S-adenosylmethionine analog by the CpG methyltransferase to a 5 carbon of a non-methylated cytosine of the double-stranded DNA so as to covalently bond the chemical group to the 5 carbon of the non- methylated cytosine of the double-stranded DNA, thereby making a derivatized double stranded DNA, wherein the chemical group has the structure :
  • R 1( R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • FIG. 1 Conversion of C to U by DNA methyltransferase (MTase) using AdoMet analog.
  • MTase transfers a chemical conversion group R from AdoMet analog to the 5 position of cytosine. After transfer, photochemically triggered intramolecular reaction between the R group and C facilitates deamination at the 4 position to form a U analog.
  • DNA MTases are able to transfer a wide variety of functional groups to the 5 position of cytosines in double stranded DNA with high sequence specificity.
  • FIG. 3 HPLC profile during time course of photo-irradiation of 5- AOMC. After 3h of irradiation, the main peak is the starting material 5-AOMC (MS-MW found 298) (Left) . After 12h photo- irradiation, the starting material is mostly consumed yielding a new product 5-AOMU with MS-MW 299 (Right) .
  • FIG. 8 Example structures of photoreactive moiety containing AdoMet analogs.
  • R 1( R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 , C(0)R', CN, N0 2 , C(0)R', S(0) 2 NHR';
  • X is 0 or NR' ;
  • R' is H or alkyl ; and
  • n l-8.
  • FIG. 9 Example scheme for the syntheses of AdoMet analogs containing photoreactive groups.
  • Figure 10. Example scheme for the syntheses of bromides of the photoreactive groups .
  • FIG. 11 DNA methylation profiling method based on DNA methyltransferase aided CpG site-specific conversion of C to U.
  • An optimized AdoMet analog is used to deliver the conversion group to an unmethylated CpG so that only modified C can be further converted to U via photo-triggered intramolecular reaction.
  • Subsequent sequencing permits DNA methylation status to be read out at single base resolution.
  • Figure 13 A. Ru (bpy) 3 2+ -visible light catalyzed photo-conversion of a 5-position modified C in DNA to a U via a cycloaddition intermediate when C is modified with an electron-deficient double bond.
  • FIG. 14 Photo-Conversion Assay. Oligonucleotides are synthesized bearing a photo-convertible 5-modified cytosine (C) on each strand within the context of the same CpG site. Prior to irradiation, or in the absence of photo-conversion to the U analog (U' ) , the site can be cut by the restriction enzyme Hpall following PCR amplification which results in replacement of the C by a normal C. After photo- conversion, PCR will convert the resulting U' to a normal U, in which case the site can be cut with Bfal. Other restriction sites are included in the DNA to produce fragment sizes that allow easy discrimination of the bands on gels or the peaks obtained with mass spectroscopy. Detailed Description of the Invention Terms
  • Nucleic acid shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof.
  • the nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA).
  • Type of nucleotide refers to A, G, C, T or U.
  • Type of base refers to adenine, guanine, cytosine, uracil or thymine.
  • Mass tag shall mean a molecular entity of a predetermined size which is capable of being attached by a cleavable bond to another entity.
  • Solid substrate shall mean any suitable medium present in the solid phase to which a nucleic acid or an agent may be affixed.
  • Non- limiting examples include chips, beads and columns.
  • Hybridize shall mean the annealing of one single-stranded nucleic acid to another nucleic acid based on sequence complementarity.
  • the propensity for hybridization between nucleic acids depends on the temperature and ionic strength of their milieu, the length of the nucleic acids and the degree of complementarity. The effect of these parameters on hybridization is well known in the art (see Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.)
  • R lt R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • R is
  • R and R 2 are independently H, alkyl, aryl, C(0)NH 2 , C(0)R',
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • Ri or R 2 is other than H.
  • R is
  • R' is H or alkyl.
  • the present invention also provides a composition of matter comprising a compound having the structure:
  • R 1( R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • R is
  • R x and R 2 are independently H, alkyl, aryl, C(0)NH 2 , C(0)R',
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • Ri or R 2 is other than H.
  • R' is H or alkyl .
  • the compound is attached to the active site of the CpG methyltransferase .
  • the CpG methyltransferase is Sssl methyltransferase .
  • the CpG methyltransferase is Hhal methyltransferase .
  • the CpG methyltransferase is CviJI methyltransferase .
  • the present invention also provides a process of producing a derivative of a double-stranded DNA comprising contacting the double-stranded DNA with a CpG methyltransferase and an S- adenosylmethionine analog having the structure:
  • R is a chemical group capable of being transferred from the S-adenosylmethionine analog by the CpG methyltransferase to a 5- carbon of a non-methylated cytosine of the double-stranded DNA, under conditions such that the chemical group covalently binds to the 5-carbon of the non-methylated cytosine of the double-stranded DNA, and thereby produces the derivative of the double-stranded DNA, wherein the chemical group has the structure:
  • R 1( R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • the chemical group has the structure
  • R x and R 2 are independently H, alkyl, aryl, C(0) H 2 , C(0)R',
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • the chemical group has the structure
  • R' is H or alkyl .
  • the CpG methyltransferase is Sssl methyltransferase . In one or more embodiments, the CpG methyltransferase is Hhal methyltransferase .
  • the CpG methyltransferase is CViJI methyltransferase .
  • the chemical group capable of being transferred from the S-adenosylmethionine analog by the CpG methyltransferase to the 5-carbon of the non-methylated cytosine of the double-stranded DNA permits photochemical deamination of a position of the non-methylated cytosine when it is covalently bound to the 5-carbon of the non-methylated cytosine of the double- stranded DNA.
  • the non-methylated cytosine is immediately adjacent in sequence to a guanine in a single strand of the double-stranded DNA.
  • the present invention also provides a method of determining whether a cytosine present within a double-stranded DNA sequence of known sequence is non-methylated comprising:
  • R is a chemical group capable of being transferred from the S-adenosylmethionine analog by the CpG methyltransferase to a 5 carbon of a non-methylated cytosine of the double-stranded DNA so as to covalently bond the chemical group to the 5 carbon of the non-methylated cytosine of the double-stranded DNA, thereby making a derivatized double stranded DNA, wherein the chemical group has the structure :
  • R lt R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 , C(0)R', CN, N0 2( C(0)R', S(0) 2 NHR'; wherein X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • step c) sequencing the single strand so obtained in step b) ; and d) comparing the sequence of the single strand determined in step c) to the sequence of a corresponding strand of the double- stranded DNA of which a derivative has not been produced, wherein the presence of a uracil analog in the single strand of the derivative single strand instead of a cytosine at a predefined position in the corresponding strand of the double- stranded DNA of which a derivative has not been produced indicates that the cytosine at that position in the double- stranded DNA is non-methylated.
  • the chemical group has the structure
  • R 2 are independently H, alkyl, aryl, C(0)NH 2 , C(0)R', CN, N0 2 , C(0)R', S(0) 2 NHR';
  • X is 0 or NR' ; wherein R' is H, alkyl or aryl; and
  • n is an integer from 1 to 8.
  • the chemical group has the structure
  • R' is H or alkyl.
  • the CpG methyltransferase is Sssl methyltransferase . In one or more embodiments, the CpG methyltransferase is Hhal methyltransferase .
  • the CpG methyltransferase is CviJI methyltransferase .
  • the non-methylated cytosine is immediately adjacent in sequence to a guanine in a single strand of the double-stranded DNA.
  • the chemical group capable of being transferred from the S-adenosylmethionine analog by the CpG methyltransferase to the 5 carbon of the non-methylated cytosine of the double-stranded DNA permits photochemical deamination of a 4 position of the non-methylated cytosine when it is covalently bound to the 5 carbon of the non-methylated cytosine of the double- stranded DNA.
  • step c) the sequencing is sequencing by synthesis.
  • the sequencing by synthesis comprises contacting the derivatized single strand with a DNA polymerase, a primer oligonucleotide, dATP, dCTP, dGTP, dTTP, and a dideoxynucleotide triphosphate having a detectable label attached thereto .
  • the detectable label is radioactive or fluorescent .
  • the detectable label is a mass tag.
  • the method further comprises attaching the single strand to a solid support prior to step c) .
  • the present invention also provides a derivatized DNA molecule, wherein the derivatized DNA molecule differs from DNA by comprising a nucleotide residue which comprises a base having the following structure : R'
  • R lf R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2 ,
  • X is 0 or NR' ' ;
  • R' ' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • R x and R 2 are independently H, alkyl, aryl, C(0)NH 2 , C(0)R'',
  • X is 0 or NR' ' ;
  • R' ' is H, alkyl or aryl
  • n is an integer from 1 to 8 ,
  • R' ' is H or alkyl.
  • the present invention also provides a derivatized DNA molecule, wherein the derivatized DNA molecule differs from DNA by comprising a nucleotide residue which comprises a base having the following
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8
  • sugar is a sugar of the nucleotide residue
  • R 1 and R 2 are independently H, alkyl, aryl, C(0)NH 2 , C(0)R',
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • R' is H or alkyl
  • the present invention also provides a kit for derivatizing a double- stranded DNA molecule or for determining whether a cytosine present within a double-stranded DNA sequence of known sequence is non- methylated comprising: a) a compound having the structure:
  • R lf R 2 and R 3 are independently H, alkyl, aryl, C(0)NH 2(
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • R is
  • Rj . and R 2 are independently H, alkyl, aryl, C(0)NH 2( C(0)R',
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • R' is H or alkyl .
  • the kit further comprises a CpG methyltransferase .
  • the CpG methyltransferase is Sssl methyltransferase . In one or more embodiments, the CpG methyltransferase is Hhal methyltransferase .
  • the CpG methyltransferase is CviJI methyltransferase .
  • the present invention also provides a method of determining whether a cytosine present within a double-stranded DNA sequence of known sequence is non-methylated comprising:
  • R is a chemical group capable of being transferred from the S-adenosylmethionine analog by the CpG methyltransferase to a 5 carbon of a non-methylated cytosine of the double-stranded DNA so as to covalently bond the chemical group to the 5 carbon of the non- methylated cytosine of the double-stranded DNA, thereby making a derivatized double stranded DNA, wherein the chemical group has the structure :
  • R 1( R 2 and R 3 are independently H, alkyl, aryl, C(0) H 2/
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • modification with the chemical group R on the cytosine at a predefined position in the double-stranded DNA indicates that the cytosine at that position in the double-stranded DNA is non- methylated.
  • the chemical group has the structure
  • R x and R 2 are independently H, alkyl, aryl, C(0)NH 2/ C(0)R',
  • X is 0 or NR' ;
  • R' is H, alkyl or aryl
  • n is an integer from 1 to 8.
  • the CpG methyltransferase is Sssl methyltransferase .
  • the CpG methyltransferase is ff al methyltransferase .
  • the CpG methyltransferase is CviJI methyltransferase .
  • the CpG methyltransferase is M. Sssl methyltransferase .
  • the CpG methyltransferase is M. ffhal methyltransferase .
  • the CpG methyltransferase is .
  • CviJI methyltransferase determining whether a cytosine at a predefined position in the double-stranded DNA has been modified with the chemical group R comprises converting the modified cytosine to a uracil analog.
  • the method further comprises conversion of a modified cysteine residue in the DNA derivative to a uracil analog by a photo-catalyzed reaction.
  • the photo-catalyzed reaction is carried out using a Tris (bipyridine) ruthenium (II) chloride (Ru(bpy) 3 2* ) catalyst .
  • the (Ru(bpy) 3 2* ) catalyst is Ru(bpy) 3 Cl 2 .
  • the (Ru(bpy) 3 2* ) catalyst is Ru (bpy) 3 (PF 6 ) 2 .
  • the light source for the photo-catalyzed reaction is a household bulb.
  • the light source for the photo-catalyzed reaction is a laser.
  • the laser has a wavelength of 400nm- 600nm.
  • This invention provides methods for methylation profiling. Methods for methylation profiling are disclosed in U.S. Patent Application Publication No. US 2011-0177508 Al, which is hereby incorporated by reference .
  • DNA methyltransferases examples include but are not limited to Sssl, Hhal and CviJI as well as modified Sssl, Hhal and CviJI (K.Sssl, M.Hhal and M. viJI , respectively). These enzymes are modified mainly to have reduced specificity such that R groups on AdoMet analogs can be more efficiently transferred to unmethylated C residues, including in the context of a CpG site in DNA. Examples of such modified M.SssI and M.Hhal genes have been described in the literature (Lukinavicius et al (2012) Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.
  • This invention also provides the instant methods and processes, wherein the DNA is bound to a solid substrate.
  • This invention also provides the instant method, wherein the DNA is bound to the solid substrate via 1,3 -dipolar azide-alkyne cycloaddition chemistry.
  • This invention also provides the instant methods and processes, wherein the DNA is bound to the solid substrate via a polyethylene glycol molecule.
  • This invention also provides the instant methods and processes, wherein the DNA is alkyne-labeled.
  • This invention also provides the instant method and processes, wherein the DNA is bound to the solid substrate via a polyethylene glycol molecule and the solid substrate is azide-functionalized.
  • This invention also provides the instant methods and processes, wherein the DNA is immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction. Immobilization of nucleic acids is described in Immobilization of DNA on Chips II, edited by Christine Wittmann (2005) , Springer Verlag, Berlin, which is hereby incorporated by reference.
  • This invention also provides the instant methods and processes, wherein the DNA is bound to the solid substrate via a polyethylene glycol molecule and the solid substrate is azide-functionalized or the DNA is immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or biotin- streptavidin interaction.
  • the DNA or nucleic acid is attached/bound to the solid surface by covalent site-specific coupling chemistry compatible with DNA.
  • This invention also provides the instant methods and processes, wherein the solid substrate is in the form of a chip, a bead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, a porous medium, or a column.
  • the solid substrate is gold, quartz, silica, plastic, glass, nylon, diamond, silver, metal, or polypropylene.
  • This invention also provides the instant method, wherein the solid substrate is porous .
  • Chips or beads may be made from materials common for DNA microarrays, for example glass or nylon. Beads/micro-beads may be in turn immobilized to chips.
  • This invention also provides the instant methods and processes, wherein about 1000 or fewer copies of the DNA are bound to the solid substrate.
  • This invention also provides the instant methods and processes wherein 2xl0 7 , lxl0 7 , lxlO 6 or lxlO 4 or fewer copies of the DNA are bound to the solid substrate.
  • nucleotide analogues comprise one of the fluorophores Cy5, Bodipy-FL-510, ROX and R6G.
  • DNA polymerase is a 9°N polymerase or a variant thereof.
  • DNA polymerases which can be used in the instant invention include, for example E.coli DNA polymerase I, Bacteriophage T4 DNA polymerase, SequenaseTM, Taq DNA polymerase and 9°N polymerase (exo-) A485L/Y409V.
  • RNA polymerases which can be used in the instant invention include, for example, Bacteriophage SP6 , T7 and T3 RNA polymerases.
  • DNA methylation at specific sequences was first analyzed by Southern blotting after cleavage with methylation-sensitive restriction endonucleases (MSREs) such as Hpall , which fails to cleave the sequence 5'-CCGG-3' when the central CpG dinucleotide is methylated (Waalwijk and Flavell, 1978) .
  • MSREs methylation-sensitive restriction endonucleases
  • Hpall methylation-sensitive restriction endonucleases
  • MSRE MSRE method is tedious, expensive, requires relatively large amounts of radioactive nucleotides, and can test only a small number of CpG sites per fragment because only -20% of all CpG sites fall within the recognition sequence of a known MSRE. If a given fragment contains many CpG sites and only one or a few are unmethylated, the sequence is often scored as unmethylated.
  • MSRE provides the best-controlled method of methylation analysis, but low throughput and other shortcomings means that it cannot form the basis for a whole-genome methylation profiling platform.
  • PCR-based methods for rapid methylation profiling of single or small numbers of CpG sites have been developed; examples are methylation-sensitive PCR (MSP; Steigerwald et al., 1990), COBRA (Eads and Laird, 2002) and methyl-light (Trinh et al . , 2001). These methods are fast and inexpensive but can test only small numbers of CpG sites; they are unsuitable for unbiased whole-genome methylation profiling. After specific methylation abnormalities have been found to be associated with a given disorder, these focused methods might be found to be appropriate for diagnostic and prognostic tests in clinical samples.
  • Microarray analysis has been applied, with considerable success (i.e., Gitan et al . , 2002).
  • microarray methods cannot address the methylation status of repeated sequences (which contain the majority of 5-methylcytosine in the genome; Rollins et al . , 2006), and CpG islands give rise to high noise levels as a result of their high G + C contents .
  • Microarrays cannot examine the methylation status of each CpG dinucleotide. Again, while this method has its advantages, it is not suited to whole-genome methylation profiling.
  • BGS bisulfite genomic sequencing
  • BGS has severe drawbacks when applied to whole genome methylation profiling.
  • the new ultrahigh throughput DNA sequencing methods cannot be used, as sequence reads are short and a large percentage of the sequences cannot be mapped to a single position in the genome. Very few repetitive sequences can be mapped at all .
  • BGS is largely restricted to pre-selected regions of the genome where primers can be designed to selectively amplify the region of interest.
  • methylation abnormalities are identified by the method of Rollins et al . (2006). It should be noted that the method disclosed herein can be applied to any sequenced genome; mammary carcinoma is shown because highly abnormal methylation patterns are known to be present in the genomes of these cells and these genomes provide an excellent test system.
  • cytosine is methylated
  • this reaction is blocked - only unmethylated CpG dinucleotides are derivatized.
  • the most important aspect of the transferred group is that it alters base pairing during sequencing or during amplification by PCR so as to allow discrimination of CpG dinucleotides that were methylated or unmethylated in the starting DNA.
  • the method is conceptually related to bisulfite genomic sequencing, but does not suffer from the deficiencies that render BGS unusable in whole-genome methylation profiling.
  • Example 1 Methods for genome-wide DNA methylation profiling based on DNA methyltransferase aided site-specific conversion of Cvtosine in CpQ islands
  • a novel genome-wide methylation profiling approach based on DNA methyltransferase aided site-specific conversion of C in unmethylated CpG dinucleotides is developed.
  • AdoMet analogs derivatized with C-reactive functionalities are used as substrates of DNA methyltransferases to transfer the reactive functionalities to the 5 position of C in unmethylated CpG dinucleotides (Fig. 1) .
  • These enzymatically attached reactive functionalities initiate highly efficient intramolecular reactions leading to conversion of C to U analogs in neutral aqueous solution.
  • high throughput DNA sequencing of the converted DNA provides a single base-resolution methylation profile; unmethylated cytosines are sequenced as thymines, while methylated cytosines are sequenced as cytosines.
  • the CpG site-specific bacterial DNA methyltransferase K-Sssl which methylates all CpG dinucleotides , transfers conversion chemical groups to the 5 position of cytosines in every unmethylated CpG dinucleotide; non-CpG cytosines and 5- methyl Cs are not modified due to the enzymes' strict CpG site recognition and high regioselectivity for the 5 position of C.
  • This has distinctive advantages over traditional bisulfite chemistry including the specific conversion of only unmethylated CpG sites, instead of all unmethylated cytosines when bisulfite is used.
  • Example 2 Photochemical conversion of 5 position-modified deoxycytidine to deoxyuridine analog.
  • the outcome for conversion of C* in DNA was detected by using single base extension-MS analysis (Fig. 7) .
  • the photo-irradiated oligonucleotide was annealed with a primer 5 ' -TGCCGCACTC-3 ' (MW 2964), then incubated with ddNTPs and DNA polymerase. With the aid of DNA polymerase, one of the 4 ddNTPs can be selectively incorporated onto the 3 ' end of the primer.
  • Photo-chemistry induced conversion of C to U should be able to direct the incorporation of ddA, resulting in an elongated primer with a MW of 3261, otherwise ddG should be incorporated giving an elongated primer of MW 3277.
  • a library of 5 position-derivatized deoxycytidxne model compounds is used to systematically screen and optimize the C-reactive functionalities that most efficiently convert C into U.
  • the model compounds are partially listed in Fig. 8.
  • AdoMet analogs used as DNA methyltransferase substrates are sulfonium derivatives of AdoHcy, in which photoactive groups (R) replace the methyl group in AdoMet (Fig. 8) .
  • Such AdoMet analogs containing photoactive groups (R) serve as substrates for CpG specific DNA methyltransferase, such as Sssl, and as a result of enzymatic reaction, these photoactive groups (R) can be transferred to the 5 position of C of unmethylated CpG dinucleotides in DNA.
  • the photoactive groups (R) include alkenes (Fig. 8, a, b, c, d, e) which forms a cyclobutane intermediate with 5,6 double bond of C upon photo-irradiation, and further result in C to U conversion.
  • the photoactive groups (R) also include alkenes modified with R 1( R 2 and R 3 , which facilitates the photoreaction.
  • R lr R 2 and R 3 are hydrogen, alkyl, aryl, amide, carboxylic acid, ester, nitro group, cyano group, aldehyde, ketone and sulfonamide.
  • Either alkyl chains or chemically cleavable structures (such as ester linkers) can be inserted between the above mentioned alkenes and the sulfur atom in AdoMet analogs.
  • the photoactive groups (R) include the above mentioned alkene conjugated butadiene moiety (Fig. 8, c) . Such butadienes are linked to the sulfur atom in AdoMet analogs by a carbon chain of various length .
  • the photoactive groups (R) include maleic anhydride and maleimide analogs (Fig. 8, f, g, h, i) which are linked to the sulfur atom in AdoMet analogs by either a saturated or double bond and containing a carbon chain of various length.
  • the photoactive groups (R) also include N, N double bond containing functionalities that can be used for photoreaction with the 5,6 double bond of C upon photo-irradiation.
  • R can be tetrazole-containing moieties (Fig. 8, j, k, 1) which are linked to the sulfur atom in AdoMet analogs by either a saturated or double bond and containing a carbon chain of various length.
  • AdoMet analogs with the desired extended side chains is carried out by regioselective S-alkylation of AdoHcy with corresponding triflates or bromides of the photoreactive moieties under mild acidic conditions.
  • a diastereomeric mixture of sulfonium is expected after alkylation of AdoHcy, and further RP—HPLC (reverse phase high performance liquid chromatography) purification is conducted to isolate the enzymatically active S-epimer for the subsequent transfer reaction.
  • Examples of the synthesis route for AdoMet analog are shown in Fig. 9.
  • Triflates or bromides of the photoreactive moieties needed for AdoMet analogs synthesis can be synthesized using commercially available starting materials as shown in Fig. 10.
  • Example 6 DNA methyIt ansferase guided transfer of C-reactive functionalities on CPG-containing DMA and subsequent site-specific C to U conversion.
  • AdoMet derivatives are designed based on the results of the experiments above and a possible library of AdoMet derivatives to be synthesized is identified. Synthesis of AdoMet analogs with the desired extended side chains is carried out following reported methods (Dalhoff et al., 2006a, 2006b) by regioselective S- alkylation of AdoHcy with corresponding triflates or bromides of the photoreactive moieties under mildly acidic conditions (Fig. 9) . A diastereomeric mixture of sulfonium is expected after alkylation of AdoHcy, and further RP-HPLC purification is used to isolate the enzymatically active S-epimer for subsequent transfer reactions.
  • a CpG site-specific DNA methyltransferase (M-Sssl) is used to transfer a photo-reactive group to the 5 position of unmethylated cytosines on both synthetic DNA and genomic DNA samples (Fig. 11) .
  • MALDI-TOF MS is used to evaluate the efficiency of reactive moiety transfer onto the DNA. Further site-specific photochemical conversion of C to U is carried out using the optimized reaction conditions obtained above. Single- base extension experiments (Fig. 7) are used to study on-DNA conversion efficiency.
  • AdoMet analogs are identified, and the conditions for both enzymatic transfer of modifying functionality and on-DNA conversion are optimized.
  • the photo-irradiation conditions are further optimized by screening for optimal wavelength, intensity and other conditions (temperature, time, buffer, pH, and auxiliary ingredients) to maximize the conversion yield and minimize possible side reactions on DNA.
  • Example 7 Combined DNA methyl-transferase-aided conversion chemistry and next-generation DNA sequencing to achieve real-world DNA methylation profiling
  • methylation patterns in real- world genomic DNA preparations are determined from the mammary carcinoma cell line MCF-7, for which we have very large amounts of methylation data.
  • DNA is purified by proteinase K digestion, phenol extraction, and dialysis against 10 mM Tris HC1, pH 7.2. DNA then is reacted with the optimal AdoMet derivative identified above and with M-Sssl (New England Biolabs, Inc.). The derivatized DNA then is subjected to high throughput DNA sequencing (Fig.
  • CpG dinucleotides in the NCBI reference sequence that appear as TpG are scored as unmethylated CpG dinucleotides in the starting DNA.
  • the required software has been developed and validated (Edwards et al., 2010) .
  • an inert "tail" from the added reactive groups may remain at the 5 position of the cytosine. This tail extends into the major groove of the DNA helix, but it is well known that modification of this position does not interfere with incorporation of nucleotides during polymerase extension, and this position has been modified in a large number of applications (Ju, et al . , 2006) including polymerase- catalyzed labeling of DNA and RNA with bulky adducts such as biotin, digoxigenin, and large fluorescent moieties. Such modifications do not markedly interfere with the efficiency or specificity of dNTP incorporation.
  • Our technology also can be used in single-molecule sequencing technologies that identify bases by electronic properties as the DNA passes through nanopore chambers.
  • AdoHcy derivatives are developed that allow accurate identification of derivatized cytosines so as to distinguish cytosine from 5 methyl cytosine. This represents a simple extension of the technology that allows the extremely fast and economical mapping of genomic methylation patterns .
  • the Methyl- seq method elaborated here provides converted DNA that can be sequenced not only by all current methods but is also perfectly suited to the new single molecule methods under development.
  • the resulting Ru(bpy) 3* complex then reduces an aryl enone to the key radical anion intermediate involved in [2+2] cycloaddition.
  • Ru*(bpy) 3 3+ can also pass an electron to an electron acceptor, the resulting Ru(bpy) 3 2* then oxidize electron-rich styrene, affording a radical cation that would undergo subsequent [2+2] cycloaddition.
  • Ru*(bpy) 3 2* turns out to be a powerful photoredox catalyst for [2+2] photocycloaddition of olefins (Ischay et al . , 2008; 2010; Du et al . , 2009) .
  • Its two-path photocatalysis mechanism makes Ru(bpy) 3 2* a versatile photocatalyst which can engage [2+2] cycloaddition of both electron-deficient and electron-rich olefins ( Fig . 12) .
  • This advantage provides us with a wide range of both electron-deficient and electron-rich olefins from which we screen and optimize the suitable double bond containing species that can be used to design and synthesize AdoMet analogs.
  • a variety of complementary double bond species modified with either electron withdrawal groups or electron donating groups can be used to derivatize AdoMet analogs.
  • the distance between these modified double bonds and the 5,6 double bond of C is also taken into consideration for a space and energy favorable intramolecular cycloaddition .
  • Fig . 13 shows two examples of Ru (bpy) /-visible light catalyzed photo-conversion of 5-position modified C to U in DNA.
  • Examples of Ru(bpy) 3 2* complex can be Ru(bpy) 3 Cl 2 and Ru (bpy) 3 (PF 6 ) 2 .
  • the visible light photocatalysis reaction mixture contains the Ru(bpy) 3 2* complex, tertiary amines (for example N, N-diisopropylethylamine (i- Pr 2 Net) ) , quaternary ammonium cations (such as MV(PF methyl viologen, or MV 2* ) , Mg 2* or Li * .
  • the light source can be ordinary household bulbs or lasers (with wavelength from 400nm-600nm) .
  • An alternative method to test the conversion of a C with its 5 position modified with a photoactive group within a synthetic single stranded DNA molecule to a U analog utilizes a synthetic double- stranded DNA molecule, which takes advantage of a simple gel-based restriction endonuclease assay.
  • Both strands of a DNA molecule of at least 50 base pairs are synthesized as oligonucleotides containing one modified C in a CpG context.
  • the C within the same CpG moiety in both strands is replaced with one of the 5 position modified photo-convertible analogs described earlier using standard phosphoramidite-based synthetic chemistry.
  • the oligonucleotides are designed in such a way that, after PCR, the resulting double-stranded DNA molecule will be cleaved with one restriction enzyme in the absence of photo- conversion and a different restriction enzyme in the presence of photo-conversion.
  • Confirmation of the photo-conversion event is via detection of the resulting restriction fragments on agarose gels, or following denaturation, the single strand fragments via MALDI-TOF mass spectrometry. Additional restriction sites allow for ease of discrimination of fragments on gels, while additional modifiable CpG sites elsewhere in the length of the synthetic DNA molecule provide further options for testing photo-conversion of multiple C analogs at various short distances from each other. An example of such an assay is shown (Fig. 14) .

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Abstract

La présente invention concerne un composé de structure I : (I) dans laquelle R1, R2 et R3 représentent, indépendamment, H, un groupe alkyle, aryle, C(O)NH2, C(O)R', CN, NO2, S(O)2NHR' ; X représente O ou NR' ; R' représente H, un groupe alkyle ou aryle ; et n représente un nombre entier de 1 à 8, sous réserve que quand R représente II (II) et que n est égal à 1, au moins l'un des R1, R2 ou R3 est différent de H.
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