Single-step purification of specific tRNAs by hydrophobic tagging

Abstract

Studies on translation frequently require large amounts of purified individual tRNAs. However, few purified tRNAs are commercially available. Individual tRNAs can be easily produced by in vitro transcription, but the lack of modifications of the tRNA transcripts may influence or impair their function [1,2]. The purification of specific tRNAs from total tRNA is a laborious process that requires several chromatographic steps [3]. Therefore, developing a simple and rapid method for purification of specific tRNA is of great importance. Here we describe a method for purification of individual tRNAs based on selective tagging of the amino group of specifically charged aminoacyl-tRNAs (aa-tRNAs) with 9-fluorenylmethylsuccinimidylcarbonat (FmocOSu) (Fig. 1), followed by a single chromatographic purification step, using reversedphase HPLC or hydrophobic interaction chromatography. The method is suitable for all tRNAs and allows up to 20-fold enrichment of a specific tRNA in less than 1 day effective working time. The materials used are readily available at low cost. We tested the procedure for two different tRNAs from Escherichia coli: tRNA and tRNA (the latter being tRNA specific for selenocysteine). As starting material, we used total tRNA, which is commercially available or can be prepared by standard procedures [4]. For the purification of tRNA, we used total tRNA from MRE 600 cells that contained approximately 3% tRNA according to charging with [C]alanine in an analytical aminoacylation assay. tRNA is a rare tRNA that is hardly detected in total tRNA (<1%). To increase the amount of tRNA in the initial tRNA preparation, we overproduced tRNA