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

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.