Universal methylation profiling methods

Abstract

This invention provides methods of derivatizing a double-stranded DNA comprising contacting double-stranded DNA with a CpG methyltransferase and an s-adenosylmethionine analog. This invention also provides methods of sequencing DNA to determine methylation patterns. This invention also provides neobases and methods of sequencing for methylation patterns using neobases.

1 . A compound having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         2 . The compound of  claim 1 , wherein R is 
       
         
           
           
               
               
           
         
       
     
     
         3 . A composition of matter comprising a compound having the structure: 
       
         
           
           
               
               
           
         
       
       attached to a CpG methyltransferase. 
     
     
         4 . The composition of matter of  claim 3 , wherein the compound is attached to the active site of the CpG methyltransferase. 
     
     
         5 . The composition of matter of  claim 4 , wherein the CpG methyltransferase is SssI methyltransferase. 
     
     
         6 . 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: 
       
         
           
           
               
               
           
         
       
       wherein 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. 
     
     
         7 . The process of  claim 6 , wherein the chemical group has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         8 . The process of  claim 7 , wherein the chemical group has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         9 . (canceled) 
     
     
         10 . The process of  claim 6 , wherein 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 oxidative 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. 
     
     
         11 . The process of  claim 6 , wherein the non-methylated cytosine is immediately adjacent in sequence to a guanine in a single strand of the double-stranded DNA. 
     
     
         12 . A method of determining whether a cytosine present within a double-stranded DNA sequence of known sequence is non-methylated comprising:
 a) producing a derivative of the double-stranded DNA by contacting the double-stranded DNA with a CpG methyltransferase and an s-adenosylmethionine analog having the structure:   
       
         
           
           
               
               
           
         
         
           wherein 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; 
         
         b) separately obtaining a single strand of the derivative of the double-stranded DNA; 
         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 thymidine 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. 
     
     
         13 . The method of  claim 12 , wherein the chemical group has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         14 . The method of  claim 13 , wherein the chemical group has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         15 - 22 . (canceled) 
     
     
         23 . 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: 
       
         
           
           
               
               
           
         
       
       and wherein the sugar is a sugar of the nucleotide residue. 
     
     
         24 . 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: 
       
         
           
           
               
               
           
         
       
       wherein the sugar is a sugar of the nucleotide residue. 
     
     
         25 . A compound having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         26 . (canceled) 
     
     
         27 . A method of derivatizing a double-stranded DNA comprising contacting double-stranded DNA with a CpG methyltransferase and an s-adenosylmethionine analog having the structure: 
       
         
           
           
               
               
           
         
       
       wherein 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 under conditions permitting the formation of a six-membered ring joining the 4 and 5 carbons of the non-methylated cytosine, thereby derivatizing the double-stranded DNA. 
     
     
         28 - 29 . (canceled) 
     
     
         30 . A method of determining whether a cytosine, in a DNA sequence of known sequence, is non-methylated comprising:
 a) derivatizing a double-stranded DNA of known sequence by contacting double-stranded DNA with a CpG methyltransferase and an s-adenosylmethionine analog having the structure:   
       
         
           
           
               
               
           
         
         
           wherein 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 under conditions permitting the formation of a neobase which comprises a six-membered ring joining the 4 and 5 carbons of the non-methylated cytosine, thereby derivatizing the double-stranded DNA; 
         
         b) obtaining a single strand of the derivatized double-stranded DNA; 
         c) sequencing the single strand; and 
         d) comparing the sequence of the single strand determined in step c) to sequence of a strand of the double-stranded DNA of known sequence, 
       
       wherein a neobase identified in the single strand in place of a cytosine at the corresponding residue position in the strand of the double-stranded DNA of known sequence indicates that the cytosine at that residue position in the double-stranded DNA of known sequence is non-methylated. 
     
     
         31 - 40 . (canceled) 
     
     
         41 . A compound having the structure: