Issue 10, 2017

Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells

Abstract

The ability to site-specifically incorporate two distinct noncanonical amino acids (ncAAs) into the proteome of a mammalian cell with high fidelity and efficiency will have many enabling applications. It would require the use of two different engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs, each suppressing a distinct nonsense codon, and which cross-react neither with each other, nor with their counterparts from the host cell. Three different aaRS/tRNA pairs have been developed so far to expand the genetic code of mammalian cells, which can be potentially combined in three unique ways to drive site-specific incorporation of two distinct ncAAs. To explore the suitability of using these combinations for suppressing two distinct nonsense codons with high fidelity and efficiency, here we systematically investigate: (1) how efficiently the three available aaRS/tRNA pairs suppress the three different nonsense codons, (2) preexisting cross-reactivities among these pairs that would compromise their simultaneous use, and (3) whether different nonsense-suppressor tRNAs exhibit unwanted suppression of non-cognate stop codons in mammalian cells. From these comprehensive analyses, two unique combinations of aaRS/tRNA pairs emerged as being suitable for high-fidelity dual nonsense suppression. We developed expression systems to validate the use of both combinations for the site-specific incorporation of two different ncAAs into proteins expressed in mammalian cells. Our work lays the foundation for developing powerful applications of dual-ncAA incorporation technology in mammalian cells, and highlights aspects of this nascent technology that need to be addressed to realize its full potential.

Graphical abstract: Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells

Supplementary files

Article information

Article type
Edge Article
Submitted
08 Jun 2017
Accepted
24 Aug 2017
First published
29 Aug 2017
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2017,8, 7211-7217

Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells

Y. Zheng, P. S. Addy, R. Mukherjee and A. Chatterjee, Chem. Sci., 2017, 8, 7211 DOI: 10.1039/C7SC02560B

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements