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 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.
Hahner
S
S
Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, D-48149 M-unster, Germany.
Lüdemann
H C
HC
Kirpekar
F
F
Nordhoff
E
E
Roepstorff
P
P
Galla
H J
HJ
Hillenkamp
F
F
D000818:Animals; D001236:Aspergillus oryzae / Q000201:enzymology; D001483:Base Sequence; D002417:Cattle; D002645:Chickens; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D010179:Pancreas / Q000201:enzymology; D010804:Physarum / Q000201:enzymology; D010944:Plants; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry*; D006163:Ribonuclease T1 / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014588:Ustilago / Q000201:enzymology
D009843:Oligoribonucleotides; D012313:RNA; D012260:Ribonucleases; D006163:Ribonuclease T1