Mass spectrometry sequencing of transfer ribonucleic acids by the comparative analysis of RNA digests (CARD) approach

Abstract

The comparative analysis of ribonucleic acid digests (CARD) approach for sequencing of transfer ribonucleic acids (tRNAs) is described. This method is enabled by the differential labeling of two tRNA populations. A set of reference tRNAs, whose complete sequences including modifications are known, are labeled with ¹⁶O during enzymatic digestion. The second (candidate) set of tRNAs, whose sequence information is desired, is labeled with ¹⁸O. By combining the two digests, digestion products that share the same sequence between the reference and candidate will appear as doublets separated by 2 Da. Sequence or modification differences between the two will generate singlets that can be further characterized to identify how the candidate sequence differs from the reference. Using CARD, ca. 80% of the tRNAs from the bacterium Citrobacter koseri can be sequenced using ribonuclease T1 with Escherichia coli tRNAs as the reference. During these studies, we also discovered a sequence error for Escherichia coli tRNA-Thr1, and use this method to confirm the correct sequence for that tRNA.