Methods of inducing site-specific cleavage of rna polynucleotides with t2 family ribonucleases
AssigneeWaters Technologies Corporation
InventorsBalasubrahmanyam Addepalli, Tatiana Johnston, Matthew A. Lauber
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Abstract
Disclosed herein are methods that provide optimal conditions for site-specific digestion and mapping of the sequence of RNA polynucleotides using T2 family endoribonucleases, such as RNase MC1 and Cusativin.
Claims26
A method of inducing site specific cleavage of one or more RNA polynucleotides, comprising contacting the one or more RNA polynucleotides with an RNase MC1 (MC1) enzyme in a solution having a pH of 6.5-9.5.
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A method of inducing site specific cleavage of one or more RNA polynucleotides, comprising contacting the one or more RNA polynucleotides with a Cusativin enzyme in a solution having a pH of 6.5-9.5.
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A method of determining the nucleic acid sequence of an RNA polynucleotide of interest, comprising:
- (a) contacting the RNA polynucleotide of interest with an MC1 enzyme in a solution having a pH of 6.5-9.5 to produce a population of RNA oligonucleotides, wherein at least two RNA oligonucleotides within the population have a nucleic acid sequence overlap of at least 1 nucleotide;
- (b) subjecting the RNA oligonucleotides to LC-MS analysis to determine the nucleic acid sequence of each of the RNA oligonucleotides within the population;
- and
- (c) aligning the nucleic acid sequences of the plurality of RNA oligonucleotides using the overlap between the at least two RNA oligonucleotides, thereby determining the nucleic acid sequence of the RNA polynucleotide of interest.
A method of determining the nucleic acid sequence of an RNA polynucleotide of interest, comprising:
- (a) contacting the RNA polynucleotide of interest with Cusativin enzyme in a solution having a pH of 6.5-9.5 to produce a population of RNA oligonucleotides, wherein at least two RNA oligonucleotides within the population have a nucleic acid sequence overlap of at least 1 nucleotide;
- (b) subjecting the RNA oligonucleotides to LC-MS analysis to determine the nucleic acid sequence of each of the RNA oligonucleotides within the population;
- and
- (c) aligning the nucleic acid sequences of the plurality of RNA oligonucleotides using the overlap between the at least two RNA oligonucleotides, thereby determining the nucleic acid sequence of the RNA polynucleotide of interest.
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Dependencies are inferred from the claim text.