Methods for Labeling a Population of RNA Molecules

Abstract

A method of labeling, and optionally enriching, for a population of target RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding a label to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated target RNA molecules in a sample by incubating the sample with labeled GTP and a capping enzyme; and (b) optionally enriching for target RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag. The label may be an oligonucleotide, which may further comprise an affinity group attached either internally or at 5′ or 3′ end of the oligonucleotide where the oligonucleotide label may be added directly, or indirectly via a reaction with a reactive group to the target RNA.

What is claimed is: 
     
         1 . A chemically capped mRNA comprising:
 a target mRNA comprising a 5′ guanosine triphosphate cap; and   an oligonucleotide that is joined by its 3′ or 5′ end to the 3′ position of the sugar of the guanosine via a linker.   
     
     
         2 . A chemically capped mRNA according to  claim 1 , wherein the linker contains a substituted or unsubstituted triazole. 
     
     
         3 . A chemically capped mRNA according to  claim 1 , wherein the oligonucleotide is selected from an oligoribonucleotide, a oligodeoxyribonucleotide, a peptide nucleic acid (PNA), a lock nucleic acid (LNA), an unlock nucleic acid (UNA), a triazole nucleic acid, phosphorothioate oligonucleotide, or a combination thereof. 
     
     
         4 . A chemically capped mRNA according to  claim 1 , wherein the oligonucleotide comprises one or more modified nucleotides. 
     
     
         5 . A chemically capped mRNA according to  claim 1 , wherein the oligonucleotide is joined to the linker by a reactive group selected from an azide, an alkyne, an amine, an active ester, a thiol, and a maleimide. 
     
     
         6 . A chemically capped mRNA according to  claim 1 , wherein the oligonucleotide is attached to the linker through an internal nucleotide or a branched spacer arm. 
     
     
         7 . A chemically capped mRNA according to  claim 1 , wherein the oligonucleotide comprises a fluorophore. 
     
     
         8 . A chemically capped mRNA according to  claim 1 , wherein the oligonucleotide comprises an affinity tag that is attached to the oligonucleotide at the 5′ position if the 3′ end is joined to the linker or the 3′ position if the 5′ end is joined to the linker. 
     
     
         9 . A chemically capped mRNA according to  claim 8 , wherein the affinity tag is selected from the group consisting of a biotin, desthiobiotin, avidin, streptavidin, protein A, maltose-binding protein, poly-histidine, HA-tag, c-myc tag, an epitope binding molecule, a self-labeling protein tag, S-tag and glutathione-S-transferase (GST). 
     
     
         10 . A chemically capped mRNA according to  claim 1 , wherein the chemically capped mRNA has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         11 . A chemically capped mRNA according to  claim 1 , wherein the chemically capped mRNA has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         12 . A method of linking an oligonucleotide to target RNA molecules, comprising:
 contacting a labeled GTP and a 5′-diphosphorylated or 5′ triphosphorylated 5′ end of the target RNA molecules in a sample, wherein the labeled GTP has the formula:   
       
         
           
           
               
               
           
         
         
           or a salt thereof to produce oligonucleotide linked target RNA molecules, wherein
 Z is H, OH, SH, NH2, a lower alkyl, a lower acyloxy, a lower alkylamine, a lower acylamine, or a halogenyl; 
 R is a linker; and 
 L comprises the oligonucleotide. 
 
         
       
     
     
         13 . A method of linking an oligonucleotide to target RNA molecules, comprising
 contacting a labeled GTP with a 5′-diphosphorylated or 5′-triphosphorylated 5′ end of the target RNA molecules in a sample by incubating the sample with a capping enzyme and the labeled GTP, wherein the label is an oligonucleotide that
 (a) is joined by its 3′ or 5′ end to the 3′ position of the sugar of the guanosine via a linker or 
 (b) is joined by an internal nucleotide or a branched spacer arm to the 3′ position of the sugar of the guanosine via a linker. 
   
     
     
         14 . A method according to  claim 13 , wherein the oligonucleotide is selected from an oligoribonucleotide, a oligodeoxyribonucleotide, a peptide nucleic acid (PNA), a lock nucleic acid (LNA), an unlock nucleic acid (UNA), a triazole nucleic acid, or a combination thereof. 
     
     
         15 . A method according to  claim 13 , wherein the oligonucleotide comprises one or more modified nucleotides. 
     
     
         16 . A method according to  claim 13 , wherein the oligonucleotide is attached to the linker through an internal nucleotide or a branched spacer arm. 
     
     
         17 . A method according to  claim 13 , wherein the oligonucleotide is attached to the linker by the oligonucleotide's 3′ end or 5′ end. 
     
     
         18 . A method according to  claim 13 , wherein the oligonucleotide comprises an affinity tag. 
     
     
         19 . A method according to  claim 18 , wherein the affinity tag is attached to the oligonucleotide (a) through an internal nucleotide or a branched spacer arm or (b) at the oligonucleotide's 5′ or 3′ end. 
     
     
         20 . A method according to  claim 18 , wherein the affinity tag is selected from the group consisting of a biotin, desthiobiotin, avidin, streptavidin, protein A, maltose-binding protein, poly-histidine, HA-tag, c-myc tag, an epitope binding molecule, a self-labeling protein tag, S-tag and glutathione-S-transferase (GST). 
     
     
         21 . A method according to  claim 13  further comprising purifying the oligonucleotide-labeled target RNA. 
     
     
         22 . A method according to  claim 21 , wherein the purifying comprises size-selection, gel electrophoresis, ion exchange, size-exclusion, phenol-chloroform extraction, alcohol precipitation, adsorption beads, isopycnic gradient, affinity beads, liquid chromatography, and hybridization. 
     
     
         23 . A method of  claim 13  further comprising sequencing the oligonucleotide-labeled target RNA. 
     
     
         24 . A method of  claim 13  further comprising enriching for the oligonucleotide-labeled target RNA and sequencing the enriched RNA. 
     
     
         25 . The method of  claim 13  further comprising sequencing the oligonucleotide-labeled target RNA. 
     
     
         26 . The method of  claim 13  further comprising enriching for the oligonucleotide-labeled target RNA and sequencing the enriched RNA. 
     
     
         27 . A method of linking an oligonucleotide to target RNA molecules, the method comprising:
 (a) contacting (i) a 3′-azido-GTP or a 3′-alkyne-GTP, (ii) a 5′-diphosphorylated or 5′-triphosphorylated 5′ end of the target RNA molecules in a sample, and (iii) a capping enzyme to produce, respectively, 3′-azido-guanosine capped target RNA molecules or 3′-alkyne-guanosine capped target RNA molecules,   (b) (i) reacting the azido of the 3′-azido-guanosine capped target RNA molecules with an oligonucleotide comprising an alkyne or (ii) reacting the alkyne of the 3′-alkyne-guanosine capped target RNA molecules with an oligonucleotide comprising an azide, in each case, to form oligonucleotide-linked target RNA molecules.   
     
     
         28 . A method of  claim 27 , wherein the reacting (b) further comprises a copper-mediated or copper-free azide-alkyne cycloaddition reaction. 
     
     
         29 . A method of  claim 28 , wherein the alkyne is a dibenzocyclooctyne or a difluorooctyne. 
     
     
         30 . A method of  claim 27  further comprising purifying the 3′-azido-guanosine capped or 3′-alkyne guanosine capped target RNA molecules prior to step (b). 
     
     
         31 . A method according to  claim 27 , wherein the oligonucleotide is selected from an oligoribonucleotide, an oligodeoxyribonucleotide, a peptide nucleic acid (PNA), a lock nucleic acid (LNA), an unlock nucleic acid (UNA), a triazole nucleic acid, or a combination thereof. 
     
     
         32 . A method according to  claim 27 , wherein the oligonucleotide comprises one or more modified nucleotides. 
     
     
         33 . A method according to  claim 27 , wherein the oligonucleotide comprises a linker. 
     
     
         34 . A method according to  claim 33 , wherein the oligonucleotide is attached to the linker through an internal nucleotide or a branched spacer arm. 
     
     
         35 . A method according to  claim 33 , wherein the oligonucleotide is attached to the linker by the oligonucleotide's 3′ end or 5′ end. 
     
     
         36 . A method according to  claim 27 , wherein the oligonucleotide comprises an affinity tag. 
     
     
         37 . A method according to  claim 36 , wherein the affinity tag is attached to the oligonucleotide (a) through an internal nucleotide or a branched spacer arm or (b) at the oligonucleotide's 5′ or 3′ end. 
     
     
         38 . A method according to  claim 36 , wherein the affinity tag is selected from the group consisting of a biotin, desthiobiotin, avidin, streptavidin, protein A, maltose-binding protein, poly-histidine, HA-tag, c-myc tag, an epitope binding molecule, a self-labeling protein tag, S-tag and glutathione-S-transferase (GST). 
     
     
         39 . A method according to  claim 27  further comprising purifying the oligonucleotide-labeled target RNA. 
     
     
         40 . A method according to  claim 39 , wherein the purifying comprises size-selection, gel electrophoresis, ion exchange, size-exclusion, phenol-chloroform extraction, alcohol precipitation, adsorption beads, isopycnic gradient, affinity beads, liquid chromatography, and hybridization. 
     
     
         41 . A method of  claim 27  further comprising sequencing the oligonucleotide-labeled target RNA. 
     
     
         42 . A method of  claim 36  further comprising enriching for the affinity-tagged, oligonucleotide-labeled target RNA and sequencing the enriched affinity-tagged, oligonucleotide-labeled target RNA. 
     
     
         43 . A method comprising sequencing the mRNA of the chemically capped mRNA of  claim 1  to produce a sequence. 
     
     
         44 . A method according to  claim 43  further comprising identifying transcriptional start sites (TSS) using the sequence of the mRNA. 
     
     
         45 . A method according to  claim 43 , wherein the sequencing comprises nanopore sequencing. 
     
     
         46 . A method comprising enriching for the chemically capped mRNA of  claim 1  and sequencing the mRNA of the chemically capped mRNA to produce a sequence. 
     
     
         47 . A method according to  claim 46 , wherein the enriching further comprises enriching for full length mRNA. 
     
     
         48 . A method according to  claim 46 , wherein the enriched RNA comprises a poly(A) tail. 
     
     
         49 . A method according to  claim 46  further comprising identifying transcriptional start sites (TSS) using the sequence of the mRNA. 
     
     
         50 . A method according to  claim 46 , wherein the sequencing comprises nanopore sequencing. 
     
     
         51 . A method comprising enriching for the chemically capped mRNA of  claim 7  and sequencing the mRNA of the chemically capped mRNA to produce a sequence. 
     
     
         52 . A method according to  claim 51 , wherein the sequencing comprises nanopore sequencing. 
     
     
         53 . A method for enriching for a population of RNA molecules in a mixture of RNAs, wherein each of the RNA molecules in the population of RNA molecules comprises a labeled guanosine at the 5′ end, the method comprising:
 contacting the mixture of RNAs with a capping enzyme and a labeled GTP to form the population of RNA molecules, wherein the labeled GTP comprises GTP linked to a label, wherein the label comprises an oligonucleotide and an affinity tag or a fluorescent tag; and 
 enriching for RNA comprising the affinity tag-labeled GTP using an affinity matrix that binds to the affinity tag to form an enriched population of RNA molecules. 
 
     
     
         54 . A method according to  claim 53  further comprising sequencing the enriched population of RNA molecules. 
     
     
         55 . A method according to  claim 53  further comprising quantifying the enriched population of RNA molecules by obtaining sequencing reads for the RNA molecules in the enriched population of RNA molecules. 
     
     
         56 . A method according to  claim 53  further comprising contacting the enriched population of RNA molecules with a reverse transcriptase to form a population of cDNA molecules corresponding to the enriched population of RNA molecules. 
     
     
         57 . A method according to  claim 56  further comprising sequencing the population of cDNA molecules. 
     
     
         58 . A method according to  claim 56  further comprising quantifying the population of cDNA molecules by obtaining sequencing reads for the cDNA molecules in the population of cDNA molecules. 
     
     
         59 . A method according to  claim 53 , wherein the mixture of RNAs comprises rRNA, tRNA, mRNA, microRNA, long non-coding RNA, and small RNA. 
     
     
         60 . A method according to  claim 53 , wherein the mixture of RNAs is a cell lysate or extract. 
     
     
         61 . A method for making cDNA comprising:
 (a) treating a sample comprising a capped, eukaryotic RNA or a capped, viral RNA with a decapping enzyme;   (b) adding an oligonucleotide-labeled guanosine monophosphate (GM P) to the 5′ end of the decapped RNA by incubating the decapped RNA with an oligonucleotide-labeled guanosine triphosphate (GTP), or a salt thereof, and a capping enzyme;   (c) optionally including a purification step to purify the oligonucleotide-labeled RNA;   (d) optionally adding a poly(A) tail or adaptor sequence in (a), (b), or (c); and   (e) reverse transcribing the RNA to produce cDNA,   wherein the oligonucleotide (i) is joined by its 3′ or 5′ end to the 3′ position of the sugar of the guanosine via a linker or (ii) is joined by an internal nucleotide or a branched spacer arm to the 3′ position of the sugar of the guanosine via a linker, and   wherein the oligonucleotide comprises a fluorophore.   
     
     
         62 . A method according to  claim 61 , wherein complementary sequence of the covalently linked oligonucleotide is incorporated in the cDNA sequence. 
     
     
         63 . A method for making cDNA comprising:
 (a) adding an oligonucleotide-labeled guanosine monophosphate (GMP) to the 5′ end of a eukaryotic, prokaryotic, or viral RNA having a 5′ diphosphate or a 5′ triphosphate by incubating the RNA with an oligonucleotide-labeled guanosine triphosphate (GTP), or a salt thereof, and a capping enzyme;   (b) optionally including a purification step to purify the oligonucleotide-labeled RNA;   (c) optionally adding a poly(A) tail or adaptor sequence in (a) or (b); and   (d) reverse transcribing the RNA to produce cDNA,   wherein the oligonucleotide (i) is joined by its 3′ or 5′ end to the 3′ position of the sugar of the guanosine via a linker or (ii) is joined by an internal nucleotide or a branched spacer arm to the 3′ position of the sugar of the guanosine via a linker, and   wherein the oligonucleotide comprises a fluorophore.