Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

Abstract

Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate-based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase-coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real-time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401-1407, 2017.

Reconstituting Natural Cell Elements in Synthetic Cells

“…[ 99 ] Other methods for endogenous tRNA removal include incubation of cell‐extract with RNase coated magnetic beads to degrade the tRNA prior to addition of synthetically synthesized tRNAs. [ 100 ]…”

Engineering molecular translation systems

“…Several approaches have now addressed this. In one approach, the entire tRNA pool can be depleted and then re-configured (Ahn et al, 2006;Salehi et al, 2017). In another, specific tRNAs can be sequestered with synthetic oligonucleotides that hybridize to the anticodon loop of tRNA (Cui et al, 2017;Cui et al, 2018).…”

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

“…Nevertheless, several methods for the replacement of endogenous tRNAs have been developed. Recently, a near-complete endogenous tRNA eliminating method was reported Bundy et al (2008) in which RNase A was attached to magnetic beads to remove native tRNAs from cell extract ( Salehi et al, 2017 ). Additionally, semisynthetic tRNA complements, which contain the majority of 48 synthetic tRNAs, were shown to be functional in CFPS, extending the ability to customize additive tRNA pools for ncAA incorporation at multiple sense codons ( Cui et al, 2015 ).…”