Nucleic acid labeling methods
View Patent ↗In one aspect of the invention, a method is provided for end-labeling RNA (total RNA, mRNA, cRNA or fragmented RNA). In one aspect of the present invention, T4 RNA ligase is used to attach a 3′-labeled AMP or CMP donor to an RNA acceptor molecule. In another embodiment, a pyrophosphate molecule 3′-AppN-3′-linker-detectable moiety is used as donor molecule. In another aspect of the present invention, a method of detecting the presence of an RNA of interest in a sample is provided, the method having the following steps: providing the sample comprising RNA which may or may not have said RNA of interest; treating the sample with a fragmenting reagent to provide RNA fragments; removing phosphate groups from said fragments to provide fragments with free 3′ OH groups; ligating said fragment with a labeling reagent according to the instant invention; providing a nucleic acid array having probes directed to said RNA of interest; hybridizing the labeled nucleic acid fragments to said nucleic acid array; and determining the extent of hybridization to said probes to determine the presence of said RNA of interest.
1. A method of detecting the presence of an RNA of interest, said method comprising the following steps:
providing a sample comprising RNA which may or may not have said RNA of interest;
ligating said RNA to a labeling reagent having the formula
wherein B is a heterocyclic moiety; X is selected from the group consisting of HO—, PO 4 2− —, P 2 O 7 3− —, P 3 O 10 4− —, OP(S)O 2 2− and adenosine-(5′)-P 2 O 7 = — having appropriate counter ions selected from the group consisting of as H + , Li + , Na + , NH4 + or K + ; Y is selected from the group consisting of —H, —OH, —OR, —SR, —NHR, or a halogen; L is a linker group selected from the group consisting of
—CH 2 —CH(OH)—CH 2 —(O—CH 2 —CH 2 ) 3 —O—CH 2 —CH 2 —CH 2 —NH— and —CH 2 —CH(OPO 3 = )—CH 2 —(O—CH 2 —CH 2 ) 3 —O—CH 2 —CH 2 —CH 2 —NH—; and Sig is a detectable moiety to provide labeled RNAs;
providing a nucleic acid array having probes directed to said RNA of interest;
hybridizing the labeled RNAs to said nucleic acid array;
and determining the extent of hybridization to said probes to determine the presence of said RNA of interest.
2. A method according to claim 1 wherein said RNA comprises microRNA.
3. A method according to claim 1 further comprising the steps of
treating said sample with a fragmenting reagent to provide RNA fragments; and
removing phosphate groups from said fragments to provide fragments with free 3′ OH groups after said step of providing said sample and before said step of ligating.
4. A method according to claim 3 wherein said fragmenting agent is selected from the group consisting of RNAse III and a buffer containing a divalent metal ion such as Mg2+ and having a pH in the neutral to alkaline range.
5. A method according to claim 3 wherein said step of removing phosphate groups from 3′ hydroxyl groups is carried out with alkaline phosphatase.
6. A method according to claim 1 wherein X is selected from the group consisting of HO—, PO 4 2− , P 2 O 7 3− —, P 3 O 10 4− —, OP(S)O 2 2− and adenosine-(5′)-P 2 O 7 = — having appropriate counter ions selected from the group consisting of as H + , Li + , Na + , NH4 + or K + .
7. A method according to claim 1 wherein Y is F.
8. A method according to claim 3 wherein said RNA comprises mRNA.
9. A method according to claim 3 wherein said RNA comprises cRNA.
10. A method according to claim 1 wherein said ligase is T4 RNA ligase.
11. A method according to claim 1 wherein Y is —OH.
12. A method according to claim 1 wherein said nucleic acid array is an oligonucleotide array.
13. A method according to claim 12 wherein said oligonucleotide array is prepared by photolithography.
14. A method according to claim 1 wherein L is —CH 2 —CH(OH)—CH 2 —(O—CH 2 —CH 2 ) 3 —O—CH 2 —CH 2 —CH 2 —NH—.
15. A method according to claim 1 wherein X is PO 4 ═.
16. A method according to claim 1 wherein B is selected from the group consisting of a pyrimidine base, a purine base, a natural base analog and an unnatural analogue.
17. A method according to claim 16 wherein B is selected from the group of the adenine, guanine, cytosine, and uracil.
18. A method according to claim 17 wherein B is selected from the group of adenine and cytosine.
19. A method according to claim 18 wherein B is cytosine.
20. A method according to claim 1 wherein said labeling reagent comprises the following structure:
wherein R is H or PO 3 2− .
21. A method according to claim 20 wherein R is H.
22. A method according to claim 1 wherein said labeling reagent comprises the following structure:
wherein R is H or PO 3 2− .
23. A method according to claim 20 wherein R is H.
24. A method according to claim 1 wherein said labeling reagent comprises the following structure:
wherein B is selected from the group consisting of adenine, guanine, cytosine, and uracil; R is H or PO 3 = .
25. A method according to claim 24 wherein B is selected from the group of adenine and cytosine and R is H.
26. A method according to claim 25 wherein B is adenine.
27. A method of claim 25 where B is cytosine.
28. A method according to claim 1 wherein said labeling reagent is selected from the group consisting of