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

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-tRNA₂ₔ₀^Opt and Ma-tRNA₂ₔ₀^Pyl, tRNAs of critical importance for genetic code expansion. We confirmed that both Mj-tRNA₂ₔ₀^Opt and Ma-tRNA₂ₔ₀^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-tRNA₂ₔ₀^Opt and Ma-tRNA₂ₔ₀^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. colitRNA₂₂₀^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.