Nucleic Acids Research, Vol 25, Issue 10 1957-1964, Copyright © 1997 by Oxford University Press
S Hahner, HC Ludemann, F Kirpekar, E Nordhoff, P Roepstorff, HJ Galla and F Hillenkamp
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.
ARTICLES
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of endonuclease digests of RNA
Institute for Medical Physics and Biophysics, University of Munster, Robert-Koch-Strasse 31, D-48149 M-unster, Germany.
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