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

Abstract

Aminoacyl-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 function in vitro or in vivo have 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.

Incorporation of multiple β2-backbones into a proteinin vivousing an orthogonal aminoacyl-tRNA synthetase

“…To assess if PylRS-like enzymes would accept β 2 -hydroxy acids as substrates, we performed in vitro tRNA acylation reactions using purified enzymes and directly analyzed the products using intact tRNA LC-HRMS (Figure 2A). 25,29 We began with Ma PylRS and compared the yields of acylated tRNA Pyl from reactions containing the known substrates (S) -α-NH 2 -N ε -Boc-Lysine ( (S) -α-ΝΗ 2 1 ) and (S) -α-OH-N ε -Boc-Lysine ( (S) -α-OH 2 ) as well as the enantiopure β 2 -OH analogs 3 and 4 . Reactions performed using 12.5 μM Ma PylRS, 25 μΜ Ma tRNA Pyl , and 10 mM 1 or 2 and incubated for 2 hours at 37°C generated the expected mono-acylated tRNA products 1 -acyl-tRNA Pyl (23188.4 Da) and 2 -acyl-tRNA Pyl (23189.6 Da) (Figure 2B , Supplementary Figure 1) in yields of 56% and 59%, respectively (Figure 2C).…”

Nonenzymatic, prebiotic aminoacylation couples chirality of RNA and protein

“…The tube was snap frozen in liquid nitrogen and stored at -80 °C. Intact tRNA LC-MS was performed as previously reported 29 . Samples were resuspended in water before analysis using intact tRNA LC-MS.…”

Chemo-ribosomal synthesis of atropisomeric and macrocyclic peptides with embedded quinolines

“…The extent of MH acylation was monitored using both acidic polyacrylamide gel electrophoresis (aPAGE) (Supplementary Fig. 2b) and intact tRNA LC-MS 39,41 (Supplementary Fig. 2c-g).…”

“…b, Yields for flexizyme-promoted acylation of MH and tRNA fMet by the DNB esters of fM and monomers 1-4. Yields were determined using intact tRNA LC-MS 39,41 . ND indicates that an acylated product was not detected.…”