MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY (MALDI)OF ENDONUCLEASE DIGESTS OF RNA/

The determination of RNA sequences using base-specific enzymatic cleav ages is a well established method, Different synthetic RNA molecules w ere analyzed for uniformity of degradation by RNase T-1, U-2, A and Ph yM under reaction conditions compatible with Matrix-Assisted Laser Des orption/Ionization Mass Spectrometry (MALDI-MS), to identify the posit ions of G, A and pyrimidine residues, In order to get a complete set o f fragments derived from cleavage at every phosphodiester bond, the sa mples were also subjected to a limited alkaline hydrolysis. Additional ly, the 5'-terminus fragments of a 49mer RNA transcript were isolated by way of 5'-biotinylation and streptavidin-coated magnetic beads (Dyn al), followed by a RNase U-2 digestion. MALDI-MS of the generated frag ments is presented as an efficient technique for a direct read out of the nucleotide sequence.