Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography-mass spectrometry

Analytical Biochemistry
Whitney M HouserPatrick A Limbach

Abstract

Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2',3'-cyclic phosphates and 3'-linear phosphates. To generate a homogeneous 3'-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3'-phosphate-containing products that could be characterized by LC-MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce (18)O labels within the 3'-phosphate of digestion products when incubated in the presence of H2(18)O, allowing prio...Continue Reading

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Citations

Apr 2, 2016·Methods : a Companion to Methods in Enzymology·Robert RossPatrick A Limbach
Nov 12, 2016·Nature Communications·Yok Hian ChionhPeter C Dedon
Jan 21, 2017·Journal of the American Society for Mass Spectrometry·Mellie June Paulines, Patrick A Limbach
Feb 13, 2019·Journal of Bacteriology·Ningxi YuPatrick A Limbach
Aug 24, 2016·The Journal of Biological Chemistry·Balasubrahmanyam Addepalli, Patrick A Limbach
Jul 22, 2017·Analytical and Bioanalytical Chemistry·Balasubrahmanyam AddepalliPatrick A Limbach

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