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

1997; Oxford University Press; Volume: 25; Issue: 10 Linguagem: Inglês

10.1093/nar/25.10.1957

1362-4962

Stefanie Hahner,

Molecular Biology Techniques and Applications

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 T 1 , U 2 , 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 U 2 digestion. MALDI-MS of the generated fragments is presented as an efficient technique for a direct read out of the nucleotide sequence.