Conditional knockout tools: Application of site-specific incorporation of unnatural amino acid via genetic code expansion in viral and parasite vaccine development

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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Evolving Bacterial Fitness with an Expanded Genetic Code

10.1101/169409 ◽

2017 ◽

Author(s):

Drew S. Tack ◽

Austin C. Cole ◽

R. Shroff ◽

B.R. Morrow ◽

Andrew D. Ellington

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acid ◽

Noncanonical Amino Acids ◽

Bacterial Fitness ◽

Genetic Code Expansion ◽

Encoding Strategy ◽

Translation Systems ◽

Expanded Genetic Code

AbstractEvolution has for the most part used the canonical 20 amino acids of the natural genetic code to construct proteins. While several theories regarding the evolution of the genetic code have been proposed, experimental exploration of these theories has largely been restricted to phylogenetic and computational modeling. The development of orthogonal translation systems has allowed noncanonical amino acids to be inserted at will into proteins. We have taken advantage of these advances to evolve bacteria to accommodate a 21 amino acid genetic code in which the amber codon ambiguously encodes either 3-nitro-L-tyrosine or stop. Such an ambiguous encoding strategy recapitulates numerous models for genetic code expansion, and we find that evolved lineages first accommodate the unnatural amino acid, and then begin to evolve on a neutral landscape where stop codons begin to appear within genes. The resultant lines represent transitional intermediates on the way to the fixation of a functional 21 amino acid code.

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Engineering RNA viruses with unnatural amino acid to evoke adjustable immune response in mice

10.1101/2021.12.04.471206 ◽

2021 ◽

Author(s):

Zhetao Zheng ◽

Yu Wang ◽

Xuesheng Wu ◽

Haoran Zhang ◽

Hongmin Chen ◽

...

Keyword(s):

Immune Response ◽

Amino Acid ◽

Genetic Code ◽

Rna Viruses ◽

Enterovirus 71 ◽

Unnatural Amino Acid ◽

Dependent Manner ◽

Strong Immune Response ◽

Genetic Code Expansion ◽

Dose Dependent

Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a model of RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro and in vivo, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and potentially protected their neonatal offspring from lethal challenge similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-tRNAPyl CUA pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.

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Sterilization of drug-resistant influenza virus through genetic interference: use of unnatural amino acid-engineered virions

10.1101/2021.12.04.471209 ◽

2021 ◽

Author(s):

Xuesheng Wu ◽

Zhetao Zheng ◽

Hongmin Chen ◽

Haishuang Lin ◽

Yuelin Yang ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Influenza A ◽

Antiviral Agent ◽

Unnatural Amino Acid ◽

Drug Resistant ◽

Genetic Code Expansion

AbstractThe frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.

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