Abstract
Over the past decade, groundbreaking discoveries have cemented transfer RNAs (tRNAs) as versatile regulators of translation and cellular function. As tRNA research gains momentum, several high-throughput sequencing methods have emerged for quantitative analysis of tRNA isoacceptors in cells. However, the strong secondary structure and rich post-transcriptional modification of most tRNA molecules pose significant challenges for reverse transcriptases, thus hampering library preparation and introducing quantification biases. Current approaches rely on processive next generation reverse transcriptases (ngRTs) - they successfully overcome these problems, albeit with the potential caveat of high experimental costs. Here, we introduce a recombinant MarathonRT (MRT) protein with a C-terminal chitin binding domain (CBD), for which we present a simple and robust one-step purification protocol that yields over 18,000 enzymatic reactions per 0.5 L of expression culture. We also developed an affordable colorimetry-based method for determining the specific activity of these enzymes. Importantly, we benchmarked our in-house produced MRT-CBD using the mim-tRNAseq workflow and show that its performance matches that of commercially available ngRTs. In addition, we implemented the use of a rapid tRNA spin column-based enrichment method for tRNA-seq and LC-MS applications, establishing it as a viable alternative to conventional gel-extraction. Combined, we provide an easily implementable MarathonRT purification protocol along with an improved workflow that significantly reduces the time and cost of tRNA-seq library preparation.