A SEQUENCING METHOD FOR RNA OLIGONUCLEOTIDES BASED ON MASS-SPECTROMETRY

Synthetic oligoribonucleotides up to 22 bases have been sequenced by o bserving different kinetics in exonuclease-induced phosphodiester bond hydrolysis and detecting the fragments by matrix-assisted laser desor ption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Comm on mass spectrometric sequencing methods have disadvantages concerning read length, cost, and specialist instrumentation using RNA as the ta rget molecule because uridine and cytidine have similar masses. Now we are in the position to distinguish U and C by different peak intensit ies. The method proposed has been verified using specific endonuclease s and C-13-labeled nucleotides. This new nongel-based and nonlabeling sequencing strategy offers first RNA sequencing data using the advanta ges of fast and accurate MALDI-TOF-MS. Preparation steps and measureme nts are performed in less than 1 h.