A NOVEL METHOD FOR THE DETERMINATION OF POSTTRANSCRIPTIONAL MODIFICATION IN RNA BY MASS-SPECTROMETRY

A method is described for the detection, chemical characterization and sequence placement of post-transcriptionally modified nucleotides in RNA. Molecular masses of oligonucleotides produced by RNase T1 hydroly sis can be measured by electrospray mass spectrometry with errors of l ess than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass. Used in conjunction with combined liquid chromatography-mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level, and assigned to sequence sites within oligon ucleotides defined by selective RNase cleavage. The procedures are dem onstrated using E. coli 5S rRNA, in which all RNase T1 fragments predi cted from the rDNA sequence are identified solely on the basis of thei r molecular masses, and using E. coli 16S rRNA for analysis of post-tr anscriptional modification, including placement of 3-methyluridine at position 1498. The principles described are generally applicable to ot her covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linki ng, or xenobiotic modification.