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<p>A critical component of genetic code expansion applications is an aminoacyl-tRNA synthetase (RS)/tRNA pair that faithfully encodes a noncanonical amino acid (ncAA) in response to a specific codon. Here we detail a procedure to select an ncAA-specific RS from a publicly available 3.2-million-member <i>Methanomethylophilus alvus</i> pyrrolysyl-RS (<i>Ma</i>PylRS) active site mutant library. Four main parts of the procedure are: (1) preparing the library for use and creating needed cell lines; (2) life and death selections that, respectively, select for functional RSs and select against RSs that incorporate canonical amino acids; (3) three fluorescence-based status checks that provide information about the efficiency and fidelity of the surviving RSs in incorporating the target ncAA; and (4) characterizing top hits to find the best ones for use in applications. The resulting RS/tRNA pairs can be used in either bacterial or eukaryotic cells to study proteins of interest. Additionally, the stability of the <i>Ma</i>PylRSs makes them useful in cell-free ncAA-protein expression and amenable to structural and other in vitro characterizations. This Protocol is usable by those with basic molecular biology expertise and features a reliable positive control scheme for selections, status checks at different stages to interpret the level of success and a robust procedure to characterize newly engineered tRNA–RS pairs. Users of this Protocol can expect to select ncAA-specific RS/tRNA pairs from the library within about 30–50 d depending on preparation needs.</p>

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Selecting aminoacyl-tRNA synthetase/tRNA pairs for efficient genetic encoding of noncanonical amino acids into proteins

  • 期刊论文

  • 发表日期：

    2025年9月22日

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  • Nathan D. Alexander,
  • Yogesh M. Gangarde,
  • Riley M. Bednar,
  • P. Andrew Karplus,
  • Richard B. Cooley,
  • Ryan A. Mehl

摘要

A critical component of genetic code expansion applications is an aminoacyl-tRNA synthetase (RS)/tRNA pair that faithfully encodes a noncanonical amino acid (ncAA) in response to a specific codon. Here we detail a procedure to select an ncAA-specific RS from a publicly available 3.2-million-member Methanomethylophilus alvus pyrrolysyl-RS (MaPylRS) active site mutant library. Four main parts of the procedure are: (1) preparing the library for use and creating needed cell lines; (2) life and death selections that, respectively, select for functional RSs and select against RSs that incorporate canonical amino acids; (3) three fluorescence-based status checks that provide information about the efficiency and fidelity of the surviving RSs in incorporating the target ncAA; and (4) characterizing top hits to find the best ones for use in applications. The resulting RS/tRNA pairs can be used in either bacterial or eukaryotic cells to study proteins of interest. Additionally, the stability of the MaPylRSs makes them useful in cell-free ncAA-protein expression and amenable to structural and other in vitro characterizations. This Protocol is usable by those with basic molecular biology expertise and features a reliable positive control scheme for selections, status checks at different stages to interpret the level of success and a robust procedure to characterize newly engineered tRNA–RS pairs. Users of this Protocol can expect to select ncAA-specific RS/tRNA pairs from the library within about 30–50 d depending on preparation needs.