Enzymatic labeling of RNA
Methods are described in which a sample containing RNA is contacted with an enzyme having an RNA ligation activity in the presence of a labeled substrate to provide labeled RNA. Methods of performing an array analysis of a labeled RNA sample are also described.
1. A method of labeling miRNA comprising:
a) heating a sample comprising a plurality of miRNAs isolated from a biological source to at least 70°C. in a solution containing at least 35% DMSO and then cooling the sample to less than 10°C.; and
b) contacting said sample with an RNA ligase in the presence of a labeled substrate under conditions sufficient to result in coupling of the labeled substrate to the miRNA in the sample to provide labeled miRNA, the conditions including a DMSO concentration in the range from 20% to 30%, wherein the labeled substrate comprises a nucleotide moiety having a terminal 3′-phosphate group and an observable label moiety attached to the nucleotide moiety via a linking group bound to the terminal 3′-phosphate group.
2. The method of claim 1 , wherein said heating a sample comprises:
heating said sample to at least 80°C. in a solution containing at least 40% DMSO and then cooling the sample to less than 50°C.
3. The method of claim 2 , wherein the duration of heating is in the range of 10 seconds to 15 minutes.
4. The method of claim 1 , wherein the nucleotide moiety is selected from a mononucleotide moiety or an oligonucleotide moiety.
5. The method of claim 1 , wherein the nucleotide moiety has a length in the range from 2 to about 50 bases.
6. The method of claim 1 , wherein the observable label moiety is selected from a chromogenic moiety, a fluorophore, a mass label, a spin label, or a radiolabel.
7. The method of claim 1 , wherein the linking group is selected from (1) a lower alkyl group; (2) a modified lower alkyl group in which one or more linkages selected from ether-, thio-, amino-, oxo-, ester-, and amido- is present; (3) a modified lower alkyl substituted with one or more groups including lower allcyl; aryl, arakyl, alkoxyl, thioalkyl, hydroxyl, amino, sulfonyl, halo; or (4) a modified lower alkyl substituted with one or more groups including lower alkyl; alkoxyl, thioalkyl, hydroxyl, amino, sulfonyl, halo, and in which one or more linkages selected from ether-, thio-, amino-, oxo-, ester-, and amido- is present.
8. The method of claim 1 , wherein the labeled substrate comprises a single observable label moiety and the labeled miRNA consists essentially of singly-labeled miRNA.
9. The method of claim 1 , wherein the labeled miRNA is at least 70% of the initial miRNA in the sample.
10. The method of claim 1 , further comprising, prior to contacting the sample with an RNA ligase, contacting the sample with an enzyme having a 5′-phosphatase activity to remove 5′-phosphate groups from the miRNA in the sample.
11. The method of claim 1 , wherein said RNA ligase is capable of coupling a species selected from the group consisting of a nucleotide having a 5′ phosphate, an oligonucleotide having a 5′ phosphate, and an RNA having a 5′ phosphate to an oligonucleotide having a 3′ hydroxyl.
12. A method of performing an array analysis comprising:
labeling miRNA in a sample comprising a plurality of miRNAs isolated from a biological source using a method according to claim 1 to provide the labeled miRNA,
contacting the labeled miRNA with an array under conditions sufficient to provide for specific binding of labeled miRNA to the array; and
interrogating the array to provide data on binding of the labeled miRNA to the array.
13. A method of labeling miRNA comprising:
a) heating a sample comprising a plurality of miRNAs isolated from a biological source to at least 70°C. in a solution containing at least 35% DMSO and then cooling the sample to less than 10°C.; and
b) contacting said sample with an RNA ligase in the presence of a labeled substrate under conditions sufficient to result in coupling of the labeled substrate to the miRNA in the sample to provide labeled miRNA, the conditions including a DMSO concentration in the range from 20% to 30%, wherein the labeled substrate has the structure (I):
Q1-Nuc-Q2-Lnk-Lbl
wherein:
Nuc is a nucleoside moiety having a 5′-terminal and a 3′-terminal;
Q1 is a 5′-phosphate group attached to the nucleoside moiety Nuc via the 5′-terminal of the nucleoside moiety,
Q2 is a 3′-phosphate group attached to the nucleoside moiety Nuc via the 3′-terminal of the nucleoside moiety;
Lnk is a linking group; and
Lbl is an observable label moiety.