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PMID: 9115363 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of endonuclease digests of RNA.

Nucleic acids research ·Vol. 25 ·No. 10 ·1997-05-15 ·Pages 1957-64

Hahner S, Lüdemann HC, Kirpekar F, Nordhoff E, Roepstorff P, Galla HJ, Hillenkamp F

Abstract

The determination of RNA sequences using base- specific enzymatic cleavages is a well established method. Different synthetic RNA molecules were analyzed for uniformity of degradation by RNase T1, U2, A and PhyM under reaction conditions compatible with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), to identify the positions of G, A and pyrimidine residues. In order to get a complete set of fragments derived from cleavage at every phosphodiester bond, the samples were also subjected to a limited alkaline hydrolysis. Additionally, the 5'-terminus fragments of a 49mer RNA transcript were isolated by way of 5'-biotinylation and streptavidin-coated magnetic beads (Dynal), followed by a RNase U2digestion. MALDI-MS of the generated fragments is presented as an efficient technique for a direct read out of the nucleotide sequence.

MeSH Terms
Animals Aspergillus oryzae/enzymology Base Sequence Cattle Chickens Liver/enzymology Molecular Sequence Data Oligoribonucleotides/chemical synthesis,chemistry Pancreas/enzymology Physarum/enzymology Plants RNA/biosynthesis,chemistry Ribonuclease T1/metabolism Ribonucleases/metabolism Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods Substrate Specificity Transcription, Genetic Ustilago/enzymology
Chemicals
Oligoribonucleotides RNA Ribonucleases Ribonuclease T1
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Hahner S
Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, D-48149 M-unster, Germany.
Lüdemann H C
Kirpekar F
Nordhoff E
Roepstorff P
Galla H J
Hillenkamp F
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/25.10.1957
Published
1997-05-15
Pages
1957-64
Language
English
Region
England
NLM ID
0411011
PMCID
PMC146684
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/25.10.1957 PMC Full Text View journal details
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