Chintan Soni, Noam Prywes, Matthew Hall, Malavika A. Nair, David F. Savage, Alanna Schepartz and Abhishek Chatterjee*,
{"title":"A Translation-Independent Directed Evolution Strategy to Engineer Aminoacyl-tRNA Synthetases","authors":"Chintan Soni, Noam Prywes, Matthew Hall, Malavika A. Nair, David F. Savage, Alanna Schepartz and Abhishek Chatterjee*, ","doi":"10.1021/acscentsci.3c01557","DOIUrl":null,"url":null,"abstract":"<p >Using directed evolution, aminoacyl-tRNA synthetases (aaRSs) have been engineered to incorporate numerous noncanonical amino acids (ncAAs). Until now, the selection of such novel aaRS mutants has relied on the expression of a selectable reporter protein. However, such translation-dependent selections are incompatible with exotic monomers that are suboptimal substrates for the ribosome. A two-step solution is needed to overcome this limitation: (A) engineering an aaRS to charge the exotic monomer, without ribosomal translation; (B) subsequent engineering of the ribosome to accept the resulting acyl-tRNA for translation. Here, we report a platform for aaRS engineering that directly selects tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the corresponding barcode-containing tRNAs from oxidative treatment designed to damage the 3′-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of the aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of the <i>Methanomethylophilus alvus</i> pyrrolysyl-tRNA synthetase from a naïve library that enables incorporation of ncAAs into proteins in living cells.</p><p >A novel approach to engineer aminoacyl-tRNA synthetases is reported that does not rely on translational readout. It will enable the selection of mutants for charging monomers that are poor substrates for the wild-type ribosome.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7000,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.3c01557","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Central Science","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acscentsci.3c01557","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
Abstract
Using directed evolution, aminoacyl-tRNA synthetases (aaRSs) have been engineered to incorporate numerous noncanonical amino acids (ncAAs). Until now, the selection of such novel aaRS mutants has relied on the expression of a selectable reporter protein. However, such translation-dependent selections are incompatible with exotic monomers that are suboptimal substrates for the ribosome. A two-step solution is needed to overcome this limitation: (A) engineering an aaRS to charge the exotic monomer, without ribosomal translation; (B) subsequent engineering of the ribosome to accept the resulting acyl-tRNA for translation. Here, we report a platform for aaRS engineering that directly selects tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the corresponding barcode-containing tRNAs from oxidative treatment designed to damage the 3′-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of the aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of the Methanomethylophilus alvus pyrrolysyl-tRNA synthetase from a naïve library that enables incorporation of ncAAs into proteins in living cells.
A novel approach to engineer aminoacyl-tRNA synthetases is reported that does not rely on translational readout. It will enable the selection of mutants for charging monomers that are poor substrates for the wild-type ribosome.
期刊介绍:
ACS Central Science publishes significant primary reports on research in chemistry and allied fields where chemical approaches are pivotal. As the first fully open-access journal by the American Chemical Society, it covers compelling and important contributions to the broad chemistry and scientific community. "Central science," a term popularized nearly 40 years ago, emphasizes chemistry's central role in connecting physical and life sciences, and fundamental sciences with applied disciplines like medicine and engineering. The journal focuses on exceptional quality articles, addressing advances in fundamental chemistry and interdisciplinary research.