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

Nucleic Acids Research
Zhenling CuiKirill Alexandrov

Abstract

Sense codon reassignment to unnatural amino acids (uAAs) represents a powerful approach for introducing novel properties into polypeptides. The main obstacle to this approach is competition between the native isoacceptor tRNA(s) and orthogonal tRNA(s) for the reassigned codon. While several chromatographic and enzymatic procedures for selective deactivation of tRNA isoacceptors in cell-free translation systems exist, they are complex and not scalable. We designed a set of tRNA antisense oligonucleotides composed of either deoxy-, ribo- or 2'-O-methyl ribonucleotides and tested their ability to efficiently complex tRNAs of choice. Methylated oligonucleotides targeting sequence between the anticodon and variable loop of tRNASerGCU displayed subnanomolar binding affinity with slow dissociation kinetics. Such oligonucleotides efficiently and selectively sequestered native tRNASerGCU directly in translation-competent Escherichia coli S30 lysate, thereby, abrogating its translational activity and liberating the AGU/AGC codons. Expression of eGFP protein from the template harboring a single reassignable AGU codon in tRNASerGCU-depleted E. coli lysate allowed its homogeneous modification with n-propargyl-l-lysine or p-azido-l-phenylala...Continue Reading

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Citations

Aug 16, 2019·Journal of Developmental Origins of Health and Disease·J B TeeP D Acott
Nov 23, 2019·Biomolecules·Thomas CaulfieldSamuel Coulbourn Flores
Jan 4, 2019·Interdisciplinary Sciences, Computational Life Sciences·Bo HuXiaoping Liu
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Nov 30, 2019·Nucleic Acids Research·Michael J HammerlingMichael C Jewett
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Oct 30, 2020·Frontiers in Bioengineering and Biotechnology·Zhenling CuiKirill Alexandrov
Oct 31, 2020·Chembiochem : a European Journal of Chemical Biology·Zhe SuiXingguo Liang
Oct 25, 2020·International Journal of Molecular Sciences·Takahito Mukai
Mar 18, 2021·Advanced Biology·Nathaniel J Gaut, Katarzyna P Adamala
Jun 18, 2021·Cell Systems·Camila KofmanMichael C Jewett

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