Work

Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems

Year: 2026

Type: article

Abstract: Transfer RNAs (tRNAs) are utilized by the ribosome to decode the nucleic acid alphabet. tRNA structure, stability, aminoacylation efficiency, and decoding efficacy are governed by their extensive post-transcriptional modifications. In most studies, individual tRNAs are generated using in vitro transcription, which produces tRNAs devoid of these critical site-specific modifications, negatively affecting translation yields and fidelity. To address this challenge, we have developed a purification method that couples tRNA overexpression to DNA hybridization-based purification. Using this approach, we produced native tRNAs from Escherichia coli in high yield and purity while retaining their complement of native post-transcriptional modifications and translational activity. We extend this technique to the purification of Mj-$tRNA_{CUA}^{Opt}$ and Ma-$tRNA_{CUA}^{Pyl}$, tRNAs of critical importance for genetic code expansion. We confirmed that both Mj-$tRNA_{CUA}^{Opt}$ and Ma-$tRNA_{CUA}^{Pyl}$ contain native E. coli post-transcriptional modifications and provide the first complete modification profiles of each. Moreover, we found that in vivo-generated Mj-$tRNA_{CUA}^{Opt}$ and Ma-$tRNA_{CUA}^{Pyl}\ $significantly outperform their in vitro-generated counterparts in amber codon suppression in cell-free translation reactions. Finally, we purified an engineered variant of E. coli$tRNA_{CCA}^{Trp}$, extending our studies to synthetic tRNAs. We present a flexible method that generates modified tRNAs in high yield and purity, addressing a critical and persistent challenge in RNA biochemistry.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gkag208