Oligonucleotide-mediated tRNA sequestration enables one-pot sense codon reassignment in vitro

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Sense codon reassignment enables incorporating unnatural amino acids (uAAs) into proteins, but native tRNA competition hinders efficiency.
• Existing methods to deactivate native tRNAs are complex and not scalable for cell-free translation.

Purpose of the Study:

• To develop a scalable and efficient method for deactivating specific native tRNAs in cell-free translation systems.
• To enable genetic code expansion by liberating codons for uAA incorporation.

Main Methods:

• Designed and synthesized tRNA antisense oligonucleotides (deoxy-, ribo-, 2'-O-methyl ribonucleotides).
• Tested oligonucleotide binding affinity and dissociation kinetics for target tRNAs (e.g., tRNASerGCU, tRNAArgCCU).
• Applied oligonucleotides in Escherichia coli S30 lysate and Leishmania tarentolae in vitro translation systems.

Main Results:

• Methylated oligonucleotides showed high binding affinity and selectively sequestered native tRNASerGCU in E. coli lysate.
• This sequestration abrogated native tRNA activity, freeing AGU/AGC codons for uAA incorporation.
• Homogeneous modification of eGFP with uAAs was achieved, and the method proved generic and species-independent.

Conclusions:

• Developed a novel, efficient, and scalable strategy using tRNA antisense oligonucleotides for genetic code reassignment.
• This approach overcomes native tRNA competition in cell-free systems, facilitating uAA incorporation.
• The method has broad applicability in synthetic biology and protein engineering.