Sequencing of Single and Double Stranded RNA Oligonucleotides by Acid Hydrolysis and MALDI Mass Spectrometry
- 18 March 2009
- journal article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 81 (8), 3173-3179
- https://doi.org/10.1021/ac900100x
Abstract
Treatment of RNA oligonucleotides with strong acids at pH 1-2 rapidly leads to hydrolysis of the phosphodiester bonds at the 5'-position of ribose. Analysis of the resulting degradation products by MALDI coupled to an Orbitrap high resolution mass spectrometer shows almost complete mass ladders from both sides of the nucleotides without interfering fragments from base losses or internal fragments. From the mass differences between adjacent peaks of a mass ladder, the sequence can be determined. Low cleavage efficiency at the termini leads to 2mers and 3mers which can be identified by MS/MS. In this way the complete sequences of different siRNA 21mer single and double strands could be verified. This simple and fast method can be applied for controlling sequences of synthetic oligomers, as well as for de-novo sequencing. Moreover, the method is applicable for localization and identification of RNA modifications as demonstrated using the examples of an oligonucleotide with phosphorothioate backbone and of one containing 2'-methoxy-ribose modifications.Keywords
This publication has 20 references indexed in Scilit:
- Gas-phase fragmentation of oligonucleotide ionsInternational Journal of Mass Spectrometry, 2004
- Matrix-assisted laser desorption/ionization mass spectrometry methods for oligodeoxynucleotides: Improvements in matrix, detection limits, quantification, and sequencingJournal of the American Society for Mass Spectrometry, 2000
- Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDI-TOFNucleic Acids Research, 1998
- A Sequencing Method for RNA Oligonucleotides Based on Mass SpectrometryAnalytical Chemistry, 1997
- Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of endonuclease digests of RNANucleic Acids Research, 1997
- Oligonucleotide Sequence and Composition Determined by Matrix-Assisted Laser Desorption/IonizationAnalytical Chemistry, 1996
- Indirect mass spectrometric methods for characterizing and sequencing oligonucleotidesMass Spectrometry Reviews, 1996
- Direct mass spectrometric sequencing of low-picomole amounts of oligodeoxynucleotides with up to 21 bases by matrix-assisted laser desorption/ionization mass spectrometryJournal of Mass Spectrometry, 1995
- Antisense DNA oligonucleotides II: The use of matrix-assisted laser desorption/ionization mass spectrometry for the sequence verification of methylphosphonate oligodeoxyribonucleotidesRapid Communications in Mass Spectrometry, 1993
- Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a powerful tool for the mass and sequence analysis of natural and modified oligonucleotidesNucleic Acids Research, 1993