In-house produced MarathonRT comparison to uMRT and Induro in tRNA sequencing library preparation

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 for quantitative analysis of tRNA isoacceptors in cells have emerged. 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), such as Induro (NEB) and uMRT (RNAConnect), to overcome these problems. Nevertheless, using these commercial enzymes comes with a relatively high cost per reaction. Here, we introduce a redesigned MarathonRT construct with added C-terminal chitin binding domain (CBD) and present a simple and robust one-step purification protocol for the in-house production of the redesigned and the original MarathonRT construct. Next, we set up an affordable colorimetry-based method for determining the specific activity of the enzyme. Our simplified method eliminates protein precipitation and yields over 26,000 enzymatic reactions per 0.5 L culture. Importantly, the in-house produced enzymes showed equal performance to established ngRTs Induro and uMRT in tRNA-seq workflow. In addition, we benchmark the use of rapid tRNA spin column-based extraction method to traditional gel-extraction using tRNA-seq and LC-MS. This improved workflow reduces the time and cost of tRNA-seq library preparation while providing an accessible MarathonRT purification protocol.