Direct, quantitative, and comprehensive analysis of tRNA acylation using intact tRNA liquid-chromatography mass-spectrometry

Abstract

AbstractAminoacyl-tRNA synthetases (aaRSs) provide the functional and essential link between the sequence of an mRNA and the protein it encodes. aaRS enzymes catalyze a two-step chemical reaction that acylates specific tRNAs with a cognate α-amino acid. In addition to their role in translation, acylated tRNAs contribute to non-ribosomal natural product biosynthesis and are implicated in multiple human diseases. From the standpoint of synthetic biology, the acylation of tRNAs with a non-canonical α-amino acid (ncAA) or more recently, a non-α-amino acid monomer (nαAA) is a critical first step in the incorporation of these monomers into proteins, where they can be used for fundamental and applied science. These endeavors all demand an understanding of aaRS activity and specificity. Although a number of methods to monitor aaRS functionin vitroorin vivohave been developed, many evaluate only the first step of the two-step reaction, require the use of radioactivity, or are slow, difficult to generalize, or both. Here we describe an LC-MS assay that rapidly, quantitatively, and directly monitors aaRS activity by detecting the intact acyl-tRNA product. After a simple tRNA acylation reaction workup, acyl- and non-acyl-tRNA molecules are resolved using ion-pairing reverse phase chromatography and their exact masses are determined using high-resolution time-of-flight mass spectrometry. The intact tRNA assay we describe is fast, simple, and quantifies reaction yields as low as 0.23%. The assay can also be employed on tRNAs acylated with flexizyme to detect products that are undetectable using standard techniques. The protocol requires basic expertise in molecular biology, mass spectrometry, and RNAse-free techniques.