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

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.