DETECTION OF MATURE SMALL RNA MOLECULES
Methods, compositions, and kits for detecting mature small RNAs are provided herein. The methods comprise ligating at least one linker to a mature small RNA in the presence of a complementary ligation template to generate a ligation product. The ligation product is a hybrid molecule comprising the linker and the mature small RNA.
1 . A method for detecting a mature small RNA, the method comprising:
a. providing a sample comprising a mature small RNA whose sequence is known;
b. ligating a 5′ linker to the 5′ end of the mature small RNA in the presence of a complementary ligation template that spans the ligation junction, whereby a ligation product is formed, the ligation product comprising the 5′ linker and the mature small RNA; and,
c. assaying the ligation product, such that the mature small RNA is detected.
2 . The method of claim 1 , wherein the mature small RNA is selected from the group consisting of a microRNA (miRNA), a short interfering RNA (siRNA), a repeat associated siRNA (rasiRNA), a transacting siRNA (tasiRNA), a Piwi interacting RNA (piRNA), and a 21-U RNA.
3 . The method of claim 1 , wherein the sample comprising a mature small RNA is selected from the group consisting of an isolated preparation of RNA, a cellular extract, an intact cell, an in vitro transcription reaction, and a chemical synthesis.
4 . The method of claim 1 , wherein the 5′ linker is selected from the group consisting of a DNA polynucleotide and a chimeric DNA-RNA polynucleotide having at least one ribonucleotide at the 3′ end.
5 . The method of claim 4 , wherein the linker further comprises a stem-loop structure, with the 5′ end forming part of the stem and the 3′ end forming a single-stranded overhang.
6 . The method of claim 1 , wherein the complementary ligation template comprises a 3′ region that hybridizes under stringent conditions to the 3′ end of the linker and a 5′ region that hybridizes under stringent conditions to the 5′ end of the mature small RNA.
7 . The method of claim 6 , wherein the ligation template is selected from the group consisting of a RNA oligonucleotide, a chimeric DNA-RNA oligonucleotide, a DNA oligonucleotide, and a DNA oligonucleotide comprising at least one PCR blocker.
8 . The method of claim 7 , wherein the PCR blocker is selected from the group consisting of a dideoxynucleotide, an amine group, a methyl group, a phosphate group, and carbon spacers.
9 . The method of claim 1 , wherein the ligation is catalyzed by a ligase, the ligase is a template-dependent ligase selected from the group consisting of T4 DNA ligase, vaccinia DNA ligase, and mammalian DNA ligases.
10 . The method of claim 1 , wherein the mature small RNA is a microRNA, the 5′ linker is a chimeric DNA-RNA polynucleotide of about 90 nucleotides that forms a stem loop structure, the complementary ligation template is an RNA oligonucleotide of about 14 nucleotides, and the ligase is T4 DNA ligase.
11 . The method of claim 1 , wherein the ligation product is assayed by a method selected from the group consisting of an amplification method and a hybridization method.
12 . The method of claim 11 , wherein the amplification method is selected from the group consisting of quantitative real-time PCR, quantitative end-point PCR, and standard PCR
13 . The method of claim 11 , wherein the hybridization method is selected from the group consisting of nucleic acid microarray, nucleic acid-coupled microsphere array, and branched DNA technology.
14 . The method of claim 12 , wherein the PCR method comprises a forward PCR primer that corresponds to a sequence of the 5′ linker and a reverse PCR primer that is complementary to a portion of the small RNA.
15 . The method of claim 14 , wherein the PCR primers comprise at least one modified nucleotide, the modified nucleotide comprises a locked nucleic acid (LNA).
16 . The method of claim 1 , wherein step (b) is performed in the presence of a plurality of complementary ligation templates, rather than a single ligation template, such that a plurality of mature small RNAs is detected.
17 . The method of claim 16 , wherein the 5′ end of each of the ligation templates is complementary to the 5′ end of a discrete mature small RNA in the plurality of mature small RNAs.
18 . A method for detecting a population of mature small RNAs selected from the group consisting of known mature small RNAs and unknown mature small RNAs, the method comprising:
a. providing a sample comprising a population of mature small RNAs;
b. ligating a first linker to one end of a mature small RNA in the population of mature small RNAs in the presence of a first semi-degenerate ligation template that spans the first ligation junction, whereby a plurality of first ligation products is formed, each first ligation product comprising a first linker and a mature small RNA;
c. ligating a second linker to the small RNA end of a ligation product in the plurality of first ligation products in the presence of a second semi-degenerate ligation template that spans the second ligation junction, whereby a plurality of second ligation products is formed, each second ligation product comprising a first linker, a mature small RNA, and a second linker; and,
d. assaying the plurality of second ligation products, such that a population of mature small RNAs is detected.
19 . The method of claim 18 , wherein steps (b) and (c) are performed simultaneously.
20 . The method of claim 18 , wherein steps (b) and (c) are performed sequentially.
21 . The method of claim 19 , wherein the second ligation products are separated from the reaction materials before step (d).
22 . The method of claim 20 , wherein the first ligation products are separated from the reaction materials before step (c), and the second ligation products are separated from the reaction materials before step (d).
23 . The method of claim 18 , wherein the sample comprising a population of mature small RNAs is selected from the group consisting of an isolated preparation of RNA, a cellular extract, and an intact cell.
24 . The method of claim 18 , wherein the first linker is a 5′ linker, the first semi-degenerate ligation template is a 5′ semi-degenerate ligation template that spans the 5′ ligation junction, the second linker is a 3′ linker, and the second semi-degenerate ligation template is a 3′ semi-degenerate ligation template that spans the 3′ ligation junction.
25 . The method of claim 18 , wherein the first linker is a 3′ linker, the first semi-degenerate ligation template is a 3′ semi-degenerate ligation template that spans the 3′ ligation junction, the second linker is a 5′ linker, and the second semi-degenerate ligation template is a 5′ semi-degenerate ligation template that spans the 5′ ligation junction.
26 . The method of claim 18 , wherein the linkers and semi-degenerate ligation templates are selected from the group consisting of a RNA oligonucleotide, a DNA oligonucleotide, and a chimeric RNA-DNA oligonucleotide.
27 . The method of claim 18 , wherein the 5′ semi-degenerate ligation template comprises a 3′ region that hybridizes under stringent conditions to the 3′ end of the 5′ linker, and a degenerate 5′ region comprising a random mix of nucleotides, such that each ligation template hybridizes with the 5′ end of a discrete small RNA in the population of small RNAs.
28 . The method of claim 18 , wherein the 3′ semi-degenerate ligation template comprises a 5′ region that hybridizes under stringent conditions to the 5′ end of the 3′ linker, and a degenerate 3′ region comprising a random mix of nucleotides, such that each ligation template hybridizes with the 3′ end of a discrete small RNA in the population of small RNAs.
29 . The method of claim 18 , wherein the 5′ and 3′ semi-degenerate ligation templates further comprise a separation tag selected from the group consisting of biotin, digoxigenin, a fluorophore, and a magnetic particle.
30 . The method of claim 18 , wherein the 31 linker comprises a 5′ phosphate group.
31 . The method of claim 18 , wherein the 5′ linker further comprises a stem-loop structure, with the 5′ end forming part of the stem and the 3′ end forming a single-stranded overhang, and the 3′ linker further comprises a stem-loop structure, with the 3′ end forming part of the stem and the 5′ end forming a single-stranded overhang.
32 . The method of claim 18 , wherein the ligation reactions are catalyzed by a ligase, the ligase is a template-dependent ligase selected from the group consisting of T4 DNA ligase, vaccinia DNA ligase, and mammalian DNA ligases.
33 . The method of claim 18 , wherein the plurality of second ligation products is assayed by converting the ligation products into DNA copies and amplifying the DNA copies.
34 . The method of claim 33 , wherein the DNA copies are generated using a method comprising a reverse transcriptase and a reverse primer that is complementary to a region of the 3′ linker.
35 . The method of claim 33 , wherein the DNA copies are amplified using a PCR method comprising a forward PCR primer that corresponds to a sequence of the 5′ linker and a reverse PCR primer that is complementary to a sequence of the 3′ linker.
36 . The method of claim 33 , wherein the DNA copies are amplified using a transcription method comprising at least one RNA polymerase, provided that the 5′ linker and the 3′ linker each further comprise a RNA polymerase promoter sequence in the proper orientation.
37 . A kit for detecting a mature small RNA whose sequence is known, the kit comprising a 5′ linker for ligating to the 5′ end of the mature small RNA, a ligation template that is complementary to the junction between the 5′ linker and the mature small RNA, a ligase, and instructions for using the kit.
38 . The kit of claim 37 , wherein the 5′ linker is selected from the group consisting of a DNA polynucleotide and a chimeric DNA-RNA polynucleotide having at least one ribonucleotide at the 3′ end.
39 . The kit of claim 38 , wherein the 5′ linker further comprises a stem-loop structure, with the 5′ end forming part of the stem and the 3′ end forming a single-stranded overhang.
40 . The kit of claim 37 , wherein the complementary ligation template comprises a 3′ region that hybridizes under stringent conditions to the 3′ end of the 5′ linker and a 5′ region that hybridizes under stringent conditions to the 5′ end of the mature small RNA
41 . The kit of claim 37 , wherein the complementary ligation template is selected from the group consisting of a RNA oligonucleotide, a chimeric DNA-RNA oligonucleotide, a DNA oligonucleotide, and a DNA oligonucleotide comprising at least one PCR blocker, wherein the PCR blocker is selected from the group consisting of a dideoxynucleotide, an amine group, a methyl group, a phosphate group, and carbon spacers.
42 . The kit of claim 37 , wherein the ligase is a template-dependent ligase selected from the group consisting of T4 DNA ligase, vaccinia DNA ligase, and mammalian DNA ligases.
43 . The kit of claim 37 further comprising a forward PCR primer that corresponds to a sequence of the 5′ linker, a reverse PCR primer that is complementary to a portion of the small RNA, and a set of reagents for quantitative PCR.
44 . The kit of claim 37 , further comprising a plurality of complementary ligation templates for the detection of a plurality of mature small RNAs whose sequences are known, wherein the 5′ end of each of the ligation templates is complementary to the 5′ end of a discrete small RNA in the plurality of mature small RNAs.
45 . A kit for detecting a population of mature small RNAs selected from the group consisting of known small RNAs and unknown small RNAs, the kit comprising a 5′ linker for ligating to the 5′ end of a mature small RNA, a 3′ linker for ligating to the 3′ end of a mature small RNA, a 5′ semi-degenerate ligation template that is complementary to the 5′ ligation junction, a 3′ semi-degenerate complementary ligation template that is complementary to the 3′ ligation junction, a ligase, and instructions for using the kit.
46 . The kit of claim 45 , wherein the 5′ semi-degenerate ligation template comprises a 3′ region that hybridizes under stringent conditions to the 3′ end of the 5′ linker, and a degenerate 5′ region comprising a random mix of nucleotides, such that each semi-degenerate ligation template hybridizes with the 5′ end of a discrete mature small RNA in the population of mature small RNAs.
47 . The kit of claim 45 , wherein the 3′ semi-degenerate ligation template comprises a 5′ region that hybridizes under stringent conditions to the 5′ end of the 3′ linker, and a degenerate 3′ end sequence comprising a random mix of nucleotides, such that each semi-degenerate ligation template hybridizes with the 3′ end of a discrete mature small RNA in the population of mature small RNAs.
48 . The kit of claim 45 , wherein the linkers and semi-degenerate ligation templates are selected from the group consisting of a RNA oligonucleotide, a DNA oligonucleotide, and a chimeric RNA-DNA oligonucleotide.
49 . The kit of claim 45 , wherein the 5′ and 3′ semi-degenerate ligation templates further comprise a separation tag selected from the group consisting of biotin, digoxigenin, a fluorophore, and a magnetic particle.
50 . The kit of claim 45 , wherein the 3′ linker comprises a 5′ phosphate group.
51 . The kit of claim 45 , wherein the 5′ linker further comprises a stem-loop structure, with the 5′ end forming part of the stem and the 3′ end forming a single-stranded overhang, and the 3′ linker further comprises a stem-loop structure, with the 3′ end forming part of the stem and the 5′ end forming a single-stranded overhang.
52 . The kit of claim 45 , wherein the ligase is a template-dependent ligase selected from the group consisting of T4 DNA ligase, vaccinia DNA ligase, and mammalian DNA ligases.
53 . The kit of claim 45 further comprising a reverse transcriptase and a reverse primer that is complementary to a sequence of the 3′ linker.
54 . The kit of claim 45 further comprising a forward PCR primer that corresponds to a sequence of the 5′ linker, a reverse PCR primer that is complementary to a portion of the 3′ linker, and a set of reagents for quantitative PCR.