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

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dc.contributor.author Addy, Partha Sarathi
dc.date.accessioned 2021-11-11T10:54:36Z
dc.date.available 2021-11-11T10:54:36Z
dc.date.issued 2017
dc.identifier.uri https://pubs.rsc.org/en/content/articlelanding/2017/sc/c7sc02560b
dc.identifier.uri http://dspace.bits-pilani.ac.in:8080/xmlui/handle/123456789/3299
dc.description.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. en_US
dc.language.iso en en_US
dc.publisher RSC en_US
dc.subject Chemistry en_US
dc.subject Amino acids en_US
dc.subject Mammalian cells en_US
dc.title Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells en_US
dc.type Article en_US


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