Systematic Evolution and Study of UAGN Decoding tRNAs in a Genomically Recoded Bacteria

Wang, Nanxi; Shang, Xin; Cerny, Ronald L.; Niu, Wei; Guo, Jiantao

doi:10.1038/srep21898

Cited by 31 publications

(52 citation statements)

References 61 publications

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“…From a collection of previously evolved tRNA UCUA , we selected three mutants (referred hereafter as tRNA UCUA -1, tRNA UCUA -2, and tRNA UCUA -3; Table S2) with varied UAGA decoding efficiency in E. coli. 12 Among them, tRNA UCUA -1 and tRNA UCUA -3 displayed the highest and the lowest UAGA decoding efficiency, respectively (Figure S1). To examine their ability to decode UAGA in mammalian cells (293T), we employed a fluorescence-based assay, where the decoding efficiency was directly linked to the expression level of an EGFP mutant (EGFP-Tyr40UAGA) of which the codon at position Tyr40 was changed to UAGA.…”

Section: Resultsmentioning

confidence: 99%

Controlling the Replication of a Genomically Recoded HIV-1 with a Functional Quadruplet Codon in Mammalian Cells

et al. 2018

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Large efforts have been devoted to genetic code engineering in the past decade, aiming for unnatural amino acid mutagenesis. Recently, an increasing number of studies were reported to employ quadruplet codons to encode unnatural amino acids. We and others have demonstrated that the quadruplet decoding efficiency could be significantly enhanced by an extensive engineering of tRNAs bearing an extra nucleotide in their anticodon loops. In this work, we report the identification of tRNA mutants derived from directed evolution to efficiently decode a UAGA quadruplet codon in mammalian cells. Intriguingly, the trend of quadruplet codon decoding efficiency among the tested tRNA variants in mammalian cells was largely the same as that in E. coli. We subsequently demonstrate the utility of quadruplet codon decoding by the construction of the first HIV-1 mutant that lacks any in-frame amber nonsense codons and can be precisely activated by the decoding of a genomically embedded UAGA codon with an unnatural amino acid. Such conditionally activatable HIV-1 mutant can likely facilitate both fundamental investigations of HIV-1 as well as vaccine developments. The use of quadruplet codon, instead of an amber nonsense codon, to control HIV-1 replication has the advantage in that the correction of a frameshift caused by a quadruplet codon is much less likely than the reversion of an amber codon back into a sense codon in HIV-1.

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Section: Resultsmentioning

confidence: 99%

Controlling the Replication of a Genomically Recoded HIV-1 with a Functional Quadruplet Codon in Mammalian Cells

et al. 2018

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“…Plasmid pRep-TAGA-tRNA UCUA was constructed as follows: The tRNA UCUA was amplified from tRNA Tyr (M. jannaschii) by overlapping PCR with primers pRep-FP1, pRep-RP1, pRep-FP2, and pRep-RP2 (Table S1). The resulting PCR product was digested with XbaI and ligated into plasmid pRepCM12b-UAGA 22 that was pretreated with the same restriction enzyme to afford pRep-TAGA-tRNA UCUA .…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…Plasmid pLei-GFP2Q-AcPheRS-tRNA UCUA was constructed as follows: The DNA fragment that contains tRNA UCUA was obtained by digestion of plasmid pRep-TAGA-tRNA UCUA with SpeI and PstI. It was subsequently ligated into plasmid pGFP UV -UCUA-wt 22 that was pretreated with the same set of restriction enzymes to afford pLei-GFP1Q-tRNA UCUA . The quadruplet codon in pLei-GFP1Q-tRNA UCUA was at position Asn149 of GFP.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…[13][14][15][16][17][18][19][20] However, many of these approaches are hampered by low ncAA incorporation efficiency. We [21][22][23] and others 24 recently reported a new approach to enhance the efficiency of quadruplet decoding, which is based on the engineering of tRNA. In the present work, we demonstrate the first example of simultaneous incorporation of two different ncAAs in a single E. coli strain using two mutually orthogonal quadruplet codons.…”

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Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons

et al. 2019

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Genetic incorporation of noncanonical amino acids has emerged as a powerful tool for the study of protein structure and function. While the three triplet nonsense codons have been widely explored, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acid mutagenesis. Here we demonstrated for the first time that two orthogonal quadruplet codons could be used to simultaneously encode two different noncanonical amino acids within a single protein in bacterial cells. To achieve this, we fine-tuned the interaction between aminoacyl-tRNA synthetase and tRNA, which afforded up to 21-fold improvement in quadruplet codon decoding efficiency. This work represents a significant step toward the use of multiple quadruplet codons for noncanonical amino acid mutagenesis. Simultaneous incorporation of two or more noncanonical amino acids is of significant importance for biological applications that can benefit from multiple unique functional groups, such as fluorescence resonance energy transfer and nuclear magnetic resonance studies, and ultimately for the synthesis of completely unnatural biopolymers as new biomaterials.

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“…(B), (C)) entails the manipulation of essential HIV‐1 protein biosynthesis through amber suppression that is precisely controlled by three mutually dependent exogenous regulatory components, a unique amber suppressor tRNA CUA (component 1) that can decode a blank codon (e.g. amber nonsense codon or a quadruplet codon) and a special aminoacyl‐tRNA synthetase (aaRS, component 2) that charges the suppressor tRNA CUA with an unnatural amino acid (unAA; component 3).…”

Section: Development Of Hiv‐1 Vaccine Using Conditionally Replicatingmentioning

confidence: 99%

Synthetic biology approach for the development of conditionally replicating HIV-1 vaccine

Wang

Yuan

Niu

et al. 2017

J. Chem. Technol. Biotechnol

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While the combined antiretroviral therapy has resulted in a significant decrease in HIV-1 related morbidity and mortality, the HIV-1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV-1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV-1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design. Among them, live-attenuated vaccines (LAVs) provided excellent protection in a non-human primate model. However, safety concerns have precluded the current version of LAVs from clinical application. As the major component of this review, two synthetic biology approaches for improving the safety of HIV-1 LAVs through controlling HIV-1 replication are discussed. Particular focus is on a novel approach that uses unnatural amino acid-mediated suppression of amber nonsense codon to generate conditionally replicating HIV-1 variants. The objective is to attract more attention towards this promising research field and to provoke creative designs and innovative utilization of the two control strategies.

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Systematic Evolution and Study of UAGN Decoding tRNAs in a Genomically Recoded Bacteria

Cited by 31 publications

References 61 publications

Controlling the Replication of a Genomically Recoded HIV-1 with a Functional Quadruplet Codon in Mammalian Cells

Controlling the Replication of a Genomically Recoded HIV-1 with a Functional Quadruplet Codon in Mammalian Cells

Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons

Synthetic biology approach for the development of conditionally replicating HIV-1 vaccine

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