Locked nucleic acid reagents for labelling nucleic acids
The present invention relates to labeling kits containing novel non-natural nucleotide monomers and to methods of making and using such compounds. The invention further relates to a method of detecting the presence of a nucleic acid, e.g., RNA, of interest in a sample, the method having the following steps: providing the sample; ligating a nucleic acid of interest with a labeling reagent according to the instant invention; providing a nucleic acid array having probes directed to the nucleic acid 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 the nucleic acid of interest.
1. A nucleic acid labelling reagent having the formula:
or an ion thereof,
wherein B is a nucleobase; R 2 is a functional group that permits attachment to a 3′ OH group of a nucleic acid; L is a linker group; and R 1 is a detectable moiety.
2. The compound according to claim 1 , wherein B is 5-methylcytosine.
3. The compound according to claim 1 , wherein R 2 is PO 4 2− , or an acid thereof.
4. The compound according to claim 1 , wherein R 1 is selected from biotin or a cyanine dye.
5. The compound according to claim 1 , wherein L is C 1-10 -alkyl amino, wherein the amino group is bound to R 1 .
6. The compound according to claim 1 , wherein L is —(CH 2 ) 6 NH—, wherein the amino group is bound to R 1 , and R 2 is PO 4 2− , or an acid thereof.
7. The compound according to claim 1 , wherein said detectable moiety is detectable by fluorescence, luminescence, optical scattering, optical absorption, radioactivity, X-ray diffraction or absorption, magnetism, or enzymatic activity, or said detectable moiety is an antibody, biotin, or digoxigenin.
8. The compound according to claim 1 , wherein R 2 is a functional group that permits attachment to a 3′ OH group of a nucleic acid by ligation.
9. The compound according to claim 1 , wherein said detectable moiety is detectable by fluorescence.
10. A nucleic acid labelling reagent having the formula:
or an ion thereof,
wherein B is 5-methylcytosine; R 2 is PO 4 2− , or an acid thereof; L is —(CH 2 ) 6 NH—, wherein the amino group is bound to R 1 ; and R 1 is a cyanine dye.
11. A kit comprising a compound of claim 1 and a ligase.
12. The kit according to claim 11 , further comprising a nucleic acid array.
13. The kit according to claim 12 , wherein said nucleic acid array comprises an LNA.
14. A method of detecting the presence of a nucleic acid of interest, said method comprising the following steps:
(a) providing a sample comprising nucleic acid that may or may not have said nucleic acid of interest;
(b) ligating said nucleic acid in said sample to a nucleic acid labeling reagent of claim 1 to provide labelled nucleic acids;
(c) providing a collection of detection probes directed to said nucleic acid of interest;
(d) contacting the labelled nucleic acids with said collection under hybridizing conditions; and
(e) determining the extent of hybridization of said labelled nucleic acids to said detection probes to determine the presence of said nucleic acid of interest.
15. The method according to claim 14 , wherein said nucleic acid of interest is RNA.
16. The method according to claim 15 , wherein said RNA comprises miRNA.
17. The method according to claim 14 , wherein T4 RNA ligase catalyzes said ligating.
18. The method according to claim 14 , wherein said collection of detection probes is a nucleic acid array.
19. The method according to claim 14 , wherein said collection of detection probes is immobilized onto one or more solid supports, where each detection probe is present at a specified location on the support.
20. The method according to claim 19 , wherein said solid support comprises a bead or a glass slide.
21. The method according to claim 20 , wherein said bead has a characteristic that provides for its identification.
22. The method according to claim 14 , wherein B is 5-methylcytosine.
23. The method according to claim 14 , wherein R 2 is PO 4 2− , or an acid thereof.
24. The method according to claim 14 , wherein R 1 is selected from biotin or a cyanine dye.
25. The method according to claim 14 , wherein L is C 1-10 -alkyl amino, wherein the amino group is bound to R 1 .
26. The method according to claim 14 , wherein L is —(CH 2 ) 6 NH—, wherein the amino group is bound to R 1 , and R 2 is PO 4 2− , or an acid thereof.
27. The method according to claim 14 , further comprising contacting said sample with a phosphatase to remove 3′ phosphate groups from said nucleic acids in said sample.
28. The method according to claim 14 , further comprising adding an adjuvant prior to said ligating.