Methods and kit for characterizing the modified base status of a transcriptome

Abstract

This invention relates to a method of characterizing the modified base status of a transcriptome, which involves contacting a transcriptome comprising one or more modified bases with an antibody specific to the modified bases under conditions effective to bind the antibody to the modified bases; isolating, from the transcriptome, a pool of RNA transcripts to which the antibody binds; and identifying isolated RNA transcripts that are present in a higher abundance in the isolated pool relative to the transcriptome, where each of the isolated RNA transcripts that are present in a higher abundance in the isolated pool together characterize the modified base status of the transcriptome. Also disclosed are a method of diagnosis or prognosis of a disease, a method of determining the effect of a treatment on modified base levels in RNA, and a kit for characterizing the modified base status of a transcriptome.

1 . (canceled) 
     
     
         2 . A method of making a library of N 6 -methyladenosine modified (m 6 A) polynucleotides for next generation sequencing, comprising:
 providing a mixture of RNA transcripts;   contacting the RNA transcripts with an antibody specific for m 6 A;   isolating RNA polynucleotides which bind to the m 6 A antibody; and   amplifying the isolated RNA to provide a library of m 6 A modified polynucleotides for next generation sequencing.   
     
     
         3 . The method of  claim 2 , wherein the RNA transcripts comprise coding and non-coding RNA transcripts. 
     
     
         4 . The method according to  claim 2 , wherein the antibody is coupled to a magnetic bead or a paramagnetic bead. 
     
     
         5 . The method according to  claim 2 , wherein providing the mixture of RNA transcripts comprises isolating the RNA transcripts by immunoprecipitation. 
     
     
         6 . The method according to  claim 2 , wherein said mixture of RNA transcripts are fragmented prior to contact with the antibody specific for m 6 A. 
     
     
         7 . The method according to  claim 6 , wherein the RNA transcripts are fragmented into fragments of about 100 nucleotides in length. 
     
     
         8 . The method according to  claim 2 , wherein providing the mixture of RNA transcripts comprises providing a mixture of RNA transcripts from a tissue. 
     
     
         9 . The method of  claim 8 , wherein providing the mixture of RNA from a tissue comprises providing the mixture of RNA from a single tissue type. 
     
     
         10 . The method according to  claim 2 , wherein providing the mixture of RNA transcripts comprises performing polysome fractionation, poly(A) purification, exome capture, ribosomal RNA-depletion, size fractionation, lincRNA enrichment, phenol-chloroform extraction, ethanol precipitation, or microRNA/small RNA isolation. 
     
     
         11 . The method of  claim 2 , wherein contacting the RNA transcripts with an antibody specific for m 6 A comprises cross-linking the antibody to the RNA transcripts. 
     
     
         12 . A method of determining the presence of N 6 -methyladenosine (m 6 A) modifications in a population of RNA transcripts in a sample, comprising:
 providing a mixture of RNA transcripts from a sample;   contacting the RNA with an antibody specific for m 6 A;   isolating the RNA which binds to the m 6 A antibody;   amplifying the isolated RNA to provide a library of polynucleotides for next generation sequencing; and   determining the nucleotide sequences of the isolated RNA in the library to determine RNA sequences which comprise a m 6 A modification.   
     
     
         13 . The method of  claim 12 , wherein the RNA transcripts comprise coding and non-coding RNA transcripts. 
     
     
         14 . The method according to  claim 12 , wherein the antibody is coupled to a magnetic bead or a paramagnetic bead. 
     
     
         15 . The method according to  claim 12 , providing the mixture of RNA transcripts comprises isolating the RNA transcripts from the sample by immunoprecipitation. 
     
     
         16 . The method according to  claim 12 , wherein said mixture of RNA transcripts are fragmented prior to contact with the antibody specific for m 6 A. 
     
     
         17 . The method according to  claim 16 , wherein the fragments are about 100 nucleotides in length. 
     
     
         18 . The method according to  claim 12 , wherein providing the mixture of RNA transcripts comprises providing a mixture of RNA transcripts from a tissue sample. 
     
     
         19 . The method of  claim 18 , wherein providing the mixture of RNA transcripts from a tissue sample comprises providing the mixture of RNA from a single tissue type. 
     
     
         20 . The method according to  claim 12 , wherein providing the mixture of RNA transcripts comprises performing polysome fractionation, poly(A) purification, exome capture, ribosomal RNA-depletion, size fractionation, lincRNA enrichment, phenol-chloroform extraction, ethanol precipitation, or microRNA/small RNA isolation. 
     
     
         21 . The method of  claim 12 , wherein contacting the RNA with an antibody specific for N 6 -methyladenosine (m 6 A) comprises cross-linking the antibody to the RNA.