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. 1997 May 15;25(10):1957–1964. doi: 10.1093/nar/25.10.1957

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

S Hahner 1, H C Lüdemann 1, F Kirpekar 1, E Nordhoff 1, P Roepstorff 1, H J Galla 1, F Hillenkamp 1
PMCID: PMC146684  PMID: 9115363

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.

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Selected References

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