Improving RNA modification mapping sequence coverage by LC-MS through a nonspecific RNase U2-E49A mutant.

ABSTRACT: We report the identification and use of a mutant of the purine selective ribonuclease RNase U2 that randomly cleaves RNA in a manner that is directly compatible with RNA modification mapping by mass spectrometry. A number of RNase U2 mutants were generated using site-saturation mutagenesis. The enzyme activity and specificity were tested using oligonucleotide substrates, which revealed an RNase U2 E49A mutant with limited specificity and a tendency to undercut RNA. Using this mutant, RNA digestion conditions were optimized to yield long, overlapping digestion products, which improve sequence coverage in RNA modification mapping experiments. The analytical utility of this mutant was demonstrated by liquid chromatography tandem mass spectrometry (LC-MS/MS) mapping of several modified RNAs wh

SUBMITTER: Solivio B 

PROVIDER: S-EPMC6214470 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature