High-throughput and single nucleotide resolution techniques for the determination of RNA post-transcriptional modifications
AssigneeUniversity of Central Florida Research Foundation, Inc.
InventorsEda Koculi, Riley Gentry
Life of this patent
Estimating the term…
Abstract
Disclosed herein are methods and associated compositions and kits for identifying nucleoside modifications. Particularly exemplified herein are methods that involve inducing base misincorporation at locations in an RNA that harbor a nucleoside modification. The resulting cDNA can then be subjected to next generation sequencing to identify mutations with respect to a reference sequence. Moreover, the type of nucleoside modification can be determined based on the substitution pattern of a given nucleoside modification.
Claims11
A method for detecting a naturally occurring RNA post-transcriptional modification in a sample, comprising:
- performing a reverse transcription reaction of an RNA in the presence of Mn2+ to generate cDNA, wherein the Mn2+ is at a concentration sufficient to induce a base misincorporation at a location of the RNA comprising a nucleoside modification;
- and
- performing next generation sequencing on the cDNA under conditions to detect the base misincorporation, and
- determining, based on the base misincorporation, one or more nucleoside modifications comprising m2G (N2-methylguanosine), m3Ψ (3-methylpseudouridine), 5-hydroxylC (5-hydroxyl cytosine), m7G (7-methylguanosine), m4Cm (N4,2′-O-dimethylcytosine), m3U (3-methyluridine), m6,6 A (N6, N6-dimethyladenosine), m1G (1-methylguanosine), m6 A (N6-methyladenosine), Cm (2′-O-methylcytidine), Um (2′-O-methyluridine), Am (2′O-methyladenosine), Gm (2′-O-methylguanosine), Ψ (pseudouridine), dihydrouridine (D) or a combination thereof;
- and, optionally, determining, based on the base misincorporation m1A (1-methyladenosine).
A method for detecting an RNA post-transcriptional modification in a sample, comprising
- identifying one or more mutations in a DNA nucleic acid sequence in the sample made as a result of subjecting a RNA to reverse transcriptase in the presence of Mn2+;
- wherein a locus of a mutation of the one or more mutations represents a nucleoside modification.
Dependencies are inferred from the claim text.