2025/08/21 by Evan M. Kalb, Russel M. Vincent, Aaron E. Engelhart +2 · 2 voices
Biochemistry, Genetics and Molecular Biology · #Cancer-related gene regulation #RNA and protein synthesis mechanisms #RNA modifications and cancer
paper · pdf · doi:10.1101/2025.08.20.671396
openalex publication_date 2025/08/21 · openalex created_date 2025/10/10 · openalex updated_date 2026/08/01
Abstract Cell-free expression systems (CFE) are flexible protein translation platforms that simplify the central dogma into an accessible reaction space. Within these systems, bulk transfer RNAs (tRNAs) are critical substrates which deliver amino acids to the elongating ribosome. For years, CFE systems were completed with commercially available tRNA isolated from E. coli MRE600. All commercial sources of tRNA have since been discontinued, jeopardizing future work in all applications of cell-free translation. Here, we address this need by repurposing previously described tRNA isolation methods to produce tRNAs suitable for CFE applications. We isolated the tRNA pools of E. coli strains A19, BL21(DE3), and Rosetta2 BL21(DE3), finding A19 tRNAs but not BL21(DE3) or Rosetta2 BL21(DE3) capable of robust in vitro translation. We determined the abundances of individual tRNAs using tRNA-seq, finding BL21(DE3) and Rosetta2 BL21(DE3) contained outsized abundances of several tRNAs, compromising translation activity. Using codon optimization strategies which align codon usage to tRNA abundance, we were able to mitigate the impact of misaligned tRNA abundances. We extended these studies to V. natriegens , a promising platform for synthetic biology and CFE. We find that neither exogenous V. natriegens tRNAs nor codon optimization are viable options to improve translation yields. Our work here highlights the importance of tRNA abundance within the context of CFE, and simultaneously addresses a critical challenge within cell-free translation.