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Simultaneous in vitro expression of minimal 21 transfer RNAs by tRNA array method.

Ryota Miyachi1, Keiko Masuda2, Yoshihiro Shimizu2

  • 1Department of Life Science, Graduate School of Arts and Science, The University of Tokyo, Meguro, Tokyo, Japan.

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|August 26, 2025
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Researchers developed a simplified method for simultaneously synthesizing all 21 essential transfer RNAs (tRNAs) in vitro. This breakthrough advances the creation of artificial cells and simplifies tRNA preparation for genetic engineering.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Transfer RNA (tRNA) is crucial for protein translation.
  • Synthesizing the minimal set of 21 tRNAs in vitro is challenging for artificial cell construction.
  • 5' and 3' end processing of tRNAs requires complex, multi-step reactions in vivo.

Purpose of the Study:

  • To develop a simplified method for simultaneous in vitro synthesis of all 21 tRNA species.
  • To overcome the obstacle of tRNA end-processing for artificial cell development.
  • To create an efficient method for preparing tRNAs for genetic engineering applications.

Main Methods:

  • Tested two 5'-end processing methods (leader and 5'-G variants).
  • Evaluated two 3'-end processing methods (direct tRNA linkage and HDVR attachment).
  • Combined methods into a 'tRNA array' approach for simultaneous expression from a single DNA template in the PURE system.

Main Results:

  • Successfully achieved simultaneous expression of all 21 tRNAs using the tRNA array method.
  • The tRNA mixture produced supported translation levels comparable to individually synthesized tRNAs for luciferase and GFP.
  • Demonstrated a simplified, unified processing strategy for multiple tRNAs.

Conclusions:

  • The tRNA array method enables efficient, simultaneous in vitro synthesis of all 21 essential tRNAs.
  • This simplified processing is a significant step toward constructing self-reproducible artificial cells.
  • The method provides a valuable tool for preparing tRNAs for genetic code engineering and synthetic biology.