An Error-Prone T7 RNA Polymerase Mutant Generated by Directed Evolution

Brakmann, Susanne; Grzeszik, Sandra

doi:10.1002/1439-7633(20010302)2:3<212::aid-cbic212>3.3.co;2-i

Cited by 9 publications

(12 citation statements)

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“…Inefficient phage replication had also been observed in a previous study with a mutator variant of T7 RNA polymerase (T7 RNAP),25 which—because of its enzymatic specificity—could not generate inheritable errors. In contrast, the replicative DNA polymerase could force the accumulation of genetic errors that would finally culminate in an error catastrophe.…”

Section: Resultsmentioning

confidence: 68%

“…The influence exerted by T7 DNAP mutator variants on the infection cycle of wild‐type bacteriophage T7 was studied by using an approach that was previously described 25. Replication of T7 phage involves the synthesis of mRNA from “early” phage genes, one of which is gene 5 (this encodes the major subunit of T7 DNAP).…”

Section: Resultsmentioning

confidence: 99%

“…Bacteriophage T7 replication assay: These experiments were adapted from an assay described earlier 25. Cultures of E. coli BLR (each harboring a specific variant‐encoding pCLT5g5Exo − plasmid) were grown in LB medium with spectinomycin and IPTG (0.5 m M ) until A 600 ≈1 was reached.…”

Section: Methodsmentioning

confidence: 99%

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Directed Evolution of an Error‐Prone T7 DNA Polymerase that Attenuates Viral Replication

et al. 2011

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Experimental evidence exists that RNA viruses replicate with extremely high mutation rates that result in significant genetic diversity. The diverse nature of viral populations allows rapid adaptation to dynamic environments, and evolution of resistances to vaccines as well as antiviral substances. For DNA viruses that replicate at much greater fidelities, as yet, neither diverse structures in the population nor their responses to increased mutation rates have been sufficiently described. By using the example of DNA bacteriophage T7, we describe the identification of virus-specific DNA polymerase variants with decreased replication fidelities, and their impact on the efficiency of the viral infection cycle.

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Section: Resultsmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 99%

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Directed Evolution of an Error‐Prone T7 DNA Polymerase that Attenuates Viral Replication

et al. 2011

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“…However, this might be offset by lower processivity (and thus yield) as well as accuracy. For T7 RNA polymerase, the error rate has been determined as about one misincorporation in 15,000 nonmodified nucleotides, i.e., for an average transcript in the range of 1500 to 3000 nucleotides there is less than one error per mRNA molecule. To our knowledge, no such studies have been performed with the mutant variants of T7 RNA polymerase, whose more flexible acceptance of building blocks might at the same time increase the number of wrongly incorporated nucleotides.…”

Section: Manufacturing Of Mrnamentioning

confidence: 99%

Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond

et al. 2015

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In recent years, the interest in using messenger RNA (mRNA) as a therapeutic means to tackle different diseases has enormously increased. This holds true not only for numerous preclinical studies, but mRNA has also entered the clinic to fight cancer. The advantages of using mRNA compared to DNA were recognized very early on, e.g., the lack of risk for genomic integration, or the expression of the encoded protein in the cytoplasm without the need to cross the nuclear membrane. However, it was generally assumed that mRNA is just not stable enough to give rise to sufficient expression of the encoded protein. Yet, an initially small group of mRNA aficionados could demonstrate that the stability of mRNA and the efficiency, by which the encoded protein is translated, can be significantly increased by selecting the right set of cis-acting structural elements (including the 5'-cap, 5'- and 3'-untranslated regions, poly(A)-tail, and modified building blocks). In parallel, significant advances in RNA packaging and delivery have been made, extending the potential for this molecule. This paved the way for further work to prove mRNA as a promising therapeutic for multiple diseases. Here, we review the developments to optimize mRNA regarding stability, translational efficiency, and immune-modulating properties to enhance its functionality and efficacy as a therapeutic. Furthermore, we summarize the current status of preclinical and clinical studies that use mRNA for cancer immunotherapy, for the expression of functional proteins as so-called transcript (or protein) replacement therapy, as well as for induction of pluripotent stem cells.

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“…This enzyme 15 transcribes the viral genes involved in virion assembly and host cell lysis (class III genes), as well as those involved in DNA replication (class II genes), including the gene for the DNA polymerase. Directed evolution studies 16 indicate that, under the appropriate pressure, the T7 RNA polymerase can evolve towards increased error rates by accepting mutations in different regions of the molecule. Therefore, mutations in the gene for the viral RNA polymerase could increase transcription error rates and lead to phenotypic mutations, some of which would occur at the TBD/thioredoxin interaction region and enable recruitment.…”

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confidence: 99%

Evidence for a role of phenotypic mutations in virus adaptation

Luzon-Hidalgo

Risso

Delgado

et al. 2020

Preprint

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Viruses repurpose the host molecular machinery for their own proliferation, block host antiviral factors and recruit host proteins for processes essential for virus propagation. Cross-species transmission requires that the virus can establish crucial interactions in the two different environments of the new and the old hosts. To explore the molecular mechanisms behind host promiscuity, we challenged a lytic phage to propagate in a host in which a protein essential for the assembly of a functional viral replisome had been modified to hinder its recruitment. The virus adapted to the engineered host without losing the capability to propagate in the original host, but no mutations that could directly explain the recruitment of the modified protein were fixed in the viral DNA genome. Adaptation, however, correlated with mutations in the gene for the viral RNA polymerase, supporting that transcription errors led to phenotypic mutations that contributed to promiscuous recruitment. Some key molecular interactions need only occur a few times per host cell to allow virus replication. Our results then support that such virus-host interactions may be mediated by mutant proteins present at very low concentrations. The possibility arises that phenotypic mutations facilitate cross-species transmission and contribute to evasion of antiviral strategies.

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An Error-Prone T7 RNA Polymerase Mutant Generated by Directed Evolution

Cited by 9 publications

References 0 publications

Directed Evolution of an Error‐Prone T7 DNA Polymerase that Attenuates Viral Replication

Directed Evolution of an Error‐Prone T7 DNA Polymerase that Attenuates Viral Replication

Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond

Evidence for a role of phenotypic mutations in virus adaptation

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