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Updated: Sep 5, 2025

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
Published on: July 10, 2020
Mapping of the Chemical Modifications of rRNAs
Jun Yang1, Peter Watzinger2, Sunny Sharma3
1Department of Cell Biology and Neurosciences, Rutgers University, Piscataway, NJ, USA.
Abstract:
Cellular RNAs, both coding and noncoding, contain several chemical modifications. Both ribose sugars and nitrogenous bases are targeted for these chemical additions. These modifications are believed to expand the topological potential of RNA molecules by bringing chemical diversity to otherwise limited repertoire. Here, using ribosomal RNA of yeast as an example, a detailed protocol for systematically mapping various chemical modifications to a single nucleotide resolution by a combination of Mung bean nuclease protection assay and RP-HPLC is provided. Molar levels are also calculated for each modification using their UV (254 nm) molar response factors that can be used for determining the amount of modifications at different residues in other RNA molecules. The chemical nature, their precise location and quantification of modifications will facilitate understanding the precise role of these chemical modifications in cellular physiology.
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