QUANTITATIVE MEASUREMENT OF DIHYDROURIDINE IN RNA USING ISOTOPE-DILUTION LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY (LC MS)/

A method has been developed for the microscale determination of 5,6-di hydrouridine, the most common post-transcriptional modification in bac terial and eukaryotic tRNA. The method is based on stable isotope dilu tion liquid chromatography-mass spectrometry (LC/MS) using [1,3-N-15(2 )]dihydrouridine and [1,3-N-15(2)]uridine as internal standards. RNA s amples were enzymatically digested to nucleosides before addition of t he internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabe led nucleosides. Sample quantities of similar to 1 pmol tRNA and 5 pmo l 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine cont ent of Escherichia coli tRNA(VGA)(Ser) and tRNA(GGU)(Thr) controls wer e measured as 2.03 and 2.84 residues/tRNA molecule, representing accur acies of 98 and 95%, Overall precision values for the analyses of E.co li tRNA(VGA)(Ser) and E.coli tRNA(GGU)(Thr), unfractionated tRNA from E.coli and 23S rRNA from E.coli were within the range 0.43-2.4%. The m ole% dihydrouridine in unfractionated tRNA and 23S rRNA from E.coli we re determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residu es/RNA molecule respectively.