Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry

Curr Protoc Nucleic Acid Chem. 2020 Sep;82(1):e113. doi: 10.1002/cpnc.113.

Abstract

This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid-phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed-phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC-MS and LC-MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC-MS is employed first to identify RNA phosphorothioate modifications. An optimized LC-MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides Basic Protocol 2: Digestion of RNA samples extracted from cells Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry.

Keywords: LC-MS/MS; RNA phosphorothioate modification; UPLC-MS; nucleic acids.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid / methods
  • Chromatography, Reverse-Phase / methods
  • Escherichia coli / genetics
  • Humans
  • Lactobacillus / genetics
  • Mass Spectrometry / methods*
  • Phosphorothioate Oligonucleotides / chemistry*
  • Phosphorothioate Oligonucleotides / isolation & purification
  • Quality Control
  • RNA / chemistry*
  • RNA / isolation & purification
  • Reference Standards
  • Solid-Phase Synthesis Techniques / methods

Substances

  • Phosphorothioate Oligonucleotides
  • RNA

Supplementary concepts

  • Lactobacillus delbrueckii subsp. lactis