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US 10,240,188 B2Granted
Detection of chemical modifications in nucleic acids
AssigneeThe University of North Carolina at Chapel Hill
InventorsKevin M. Weeks, Nathan Siegfried, Philip Homan, Steven Busan, Oleg V. Favorov
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Abstract
The presently disclosed subject matter relates to technology and methods for analyzing the structure of nucleic acid molecules, such as RNA molecules. More particularly, the presently disclosed subject matter is directed to methods of compositions for, and computer program products for nucleic acid analysis, such as RNA structure analysis.
Claims14
A method for detecting structural data in a ribonucleic acid (RNA), the method comprising:
- a. providing a RNA;
- b. exposing the RNA to a reagent that modifies nucleotides in the RNA to form a covalent ribose 2′-O-adduct or to form a methyl adduct at the N1 position of adenosine or the N3 position of cytosine, thereby providing a modified RNA;
- c. synthesizing a deoxyribonucleic acid (DNA) using a Moloney murine leukemia virus (MMLV) reverse transcriptase and the modified RNA provided in step (b) as a template, wherein the synthesizing occurs under conditions wherein the reverse transcriptase reads through the chemical modification in the modified RNA provided in step (b) to thereby produce a noncomplementary deoxyribonucleotide or a deletion in the resulting DNA at the site of the chemical modification, wherein said conditions comprise contacting the modified RNA with the reverse transcriptase and Mn2+ for at least one hour;
- d. detecting the non-complementary deoxyribonucleotide or the deletion;
- and
- e. determining structural data for the RNA provided in step (a).
3 more elements
Dependencies are inferred from the claim text.