{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T18:03:53Z","timestamp":1784570633997,"version":"3.55.0"},"reference-count":32,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T00:00:00Z","timestamp":1784505600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"EU","award":["Horizon 2020 research and innovation programme No. 965135"],"award-info":[{"award-number":["Horizon 2020 research and innovation programme No. 965135"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Bacteriophage T7 RNA polymerase (T7 RNAP) is a key enzyme for\n                    <jats:italic>in vitro<\/jats:italic>\n                    transcription (IVT) and plays a central role in the production of synthetic mRNA for research and therapeutic applications. However, IVT frequently generates double-stranded RNA (dsRNA) as an undesired by-product, which can trigger innate immune responses and compromise mRNA quality. Increasing reaction temperatures reduces dsRNA formation, however, the wild-type T7 RNA polymerase exhibits limited stability under such conditions. Therefore, polymerase variants with enhanced thermotolerance enable more robust transcription at elevated temperatures while minimizing dsRNA generation. To address this limitation, we aimed at obtaining T7 RNA polymerase variants with increased thermotolerance using the Protein Repair One Stop Shop (PROSS) web server. Four crystal structures of T7 RNA polymerase, comprising a promoter complex, an initiation complex, and two elongation complexes were used as input for independent PROSS runs. Mutations shared across all four designs for each PROSS index were then combined to generate multi-structure PROSS Combined Designs (PCDs). In the subset evaluated experimentally, PCD9 retained full-length transcription activity at temperatures up to 48 \u00b0C, whereas wild-type T7 RNA polymerase showed strong loss of activity under the same buffer conditions. At 48 \u00b0C, PCD9 supported the synthesis of kilobase-length scale transcripts and produced no detectable dsRNA signal in a dot blot assay. In contrast, the wild-type enzyme generated strong dsRNA signals at 37 \u00b0C and failed to produce detectable RNA at 48 \u00b0C. Additional PROSS variants derived exclusively from the elongation complex structure were inactive at both 37 \u00b0C and 48 \u00b0C. Together, these results show that multi-structure PROSS design can yield a thermotolerant T7 RNA polymerase with improved performance at elevated temperature and reduced dsRNA byproduct formation. The findings also suggest that restricting stability design to a single structural state may not fully capture the requirements of a highly dynamic enzyme.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0353775","type":"journal-article","created":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T17:28:46Z","timestamp":1784568526000},"page":"e0353775","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering of T7 DNA-dependent RNA polymerase with activity at elevated 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Biol"},{"issue":"1","key":"pone.0353775.ref002","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/0022-2836(86)90385-2","article-title":"Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes","volume":"189","author":"FW Studier","year":"1986","journal-title":"J Mol Biol"},{"key":"pone.0353775.ref003","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.procbio.2024.10.005","article-title":"Purification of T7 RNA polymerase for large scale production of mRNA vaccines and therapeutics","volume":"147","author":"N Raghuwanshi","year":"2024","journal-title":"Process Biochemistry"},{"issue":"8","key":"pone.0353775.ref004","doi-asserted-by":"crossref","first-page":"2129","DOI":"10.1016\/j.biotechadv.2018.10.001","article-title":"Bacteriophage T7 transcription system: an enabling tool in synthetic biology","volume":"36","author":"W Wang","year":"2018","journal-title":"Biotechnol Adv"},{"issue":"11","key":"pone.0353775.ref005","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1093\/protein\/gzt040","article-title":"Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase","volume":"26","author":"J-C Boulain","year":"2013","journal-title":"Protein Eng Des Sel"},{"key":"pone.0353775.ref006","doi-asserted-by":"crossref","first-page":"1356354","DOI":"10.3389\/fbioe.2024.1356354","article-title":"Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst","volume":"12","author":"W He","year":"2024","journal-title":"Front Bioeng Biotechnol"},{"issue":"3","key":"pone.0353775.ref007","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1261\/rna.073858.119","article-title":"Synthesis of low immunogenicity RNA with high-temperature in vitro transcription","volume":"26","author":"MZ Wu","year":"2020","journal-title":"RNA"},{"issue":"4","key":"pone.0353775.ref008","doi-asserted-by":"crossref","first-page":"560","DOI":"10.1038\/s41587-022-01525-6","article-title":"An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts","volume":"41","author":"A Dousis","year":"2023","journal-title":"Nat Biotechnol"},{"issue":"18","key":"pone.0353775.ref009","doi-asserted-by":"crossref","first-page":"9253","DOI":"10.1093\/nar\/gky796","article-title":"3\u2019 end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses","volume":"46","author":"Y Gholamalipour","year":"2018","journal-title":"Nucleic Acids Res"},{"issue":"10","key":"pone.0353775.ref010","doi-asserted-by":"crossref","first-page":"5239","DOI":"10.1093\/nar\/gky177","article-title":"An origin of the immunogenicity of in vitro transcribed RNA","volume":"46","author":"X Mu","year":"2018","journal-title":"Nucleic Acids Res"},{"issue":"21","key":"pone.0353775.ref011","doi-asserted-by":"crossref","first-page":"4012","DOI":"10.1021\/acs.accounts.1c00521","article-title":"Immunogenicity of in vitro-transcribed RNA","volume":"54","author":"X Mu","year":"2021","journal-title":"Acc Chem Res"},{"issue":"20","key":"pone.0353775.ref012","doi-asserted-by":"crossref","first-page":"5357","DOI":"10.1093\/nar\/20.20.5357","article-title":"Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons","volume":"20","author":"M Puttaraju","year":"1992","journal-title":"Nucleic Acids Res"},{"key":"pone.0353775.ref013","doi-asserted-by":"crossref","first-page":"787881","DOI":"10.3389\/fbioe.2021.787881","article-title":"Circular RNA: biosynthesis in vitro","volume":"9","author":"X Chen","year":"2021","journal-title":"Front Bioeng Biotechnol"},{"issue":"1","key":"pone.0353775.ref014","doi-asserted-by":"crossref","first-page":"2629","DOI":"10.1038\/s41467-018-05096-6","article-title":"Engineering circular RNA for potent and stable translation in eukaryotic cells","volume":"9","author":"RA Wesselhoeft","year":"2018","journal-title":"Nat Commun"},{"issue":"6313","key":"pone.0353775.ref015","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1038\/350091a0","article-title":"Nucleic acid sequence-based amplification","volume":"350","author":"J Compton","year":"1991","journal-title":"Nature"},{"issue":"4","key":"pone.0353775.ref016","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1128\/CMR.5.4.370","article-title":"Advances in nucleic acid-based detection methods","volume":"5","author":"MJ Wolcott","year":"1992","journal-title":"Clin Microbiol Rev"},{"issue":"35","key":"pone.0353775.ref017","doi-asserted-by":"crossref","first-page":"9115","DOI":"10.1021\/bi00086a016","article-title":"Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences","volume":"32","author":"RA Ikeda","year":"1993","journal-title":"Biochemistry"},{"key":"pone.0353775.ref018","unstructured":"Liao H, Van Gemen B, Sugiyama A. Mutants of RNA polymerases with increased stability. WO2000036112A1. 2000."},{"key":"pone.0353775.ref019","unstructured":"Sugiyama A, Nishiya Y, Kawakami B. RNA polymerase mutants with increased thermostability. WO2001066705A1. 2001."},{"key":"pone.0353775.ref020","unstructured":"Sobek H. T7 RNA polymerase variants with enhanced thermostability. US20110256589A1. 2011."},{"key":"pone.0353775.ref021","unstructured":"Ong J, Chong S, Asahara H, Hung KC, Potapov V, Tzertzinis G. Thermostable variants of T7 RNA polymerase. EP3402881B1. 2021."},{"key":"pone.0353775.ref022","unstructured":"Jain R. T7 RNA polymerase variants. WO2019199807A1. 2019."},{"key":"pone.0353775.ref023","unstructured":"Sugiyama A, Nishiya Y, Kawakami B. RNA polymerase mutants with increased thermostability. US7507567B2. 2009."},{"issue":"15","key":"pone.0353775.ref024","doi-asserted-by":"crossref","first-page":"7480","DOI":"10.1093\/nar\/gkv734","article-title":"Transcription yield of fully 2\u2019-modified RNA can be increased by the addition of thermostabilizing mutations to T7 RNA polymerase mutants","volume":"43","author":"AJ Meyer","year":"2015","journal-title":"Nucleic Acids Res"},{"issue":"2","key":"pone.0353775.ref025","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/j.molcel.2016.06.012","article-title":"Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability","volume":"63","author":"A Goldenzweig","year":"2016","journal-title":"Mol Cell"},{"issue":"6731","key":"pone.0353775.ref026","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1038\/19999","article-title":"Structural basis for initiation of transcription from an RNA polymerase-promoter complex","volume":"399","author":"GM Cheetham","year":"1999","journal-title":"Nature"},{"issue":"5448","key":"pone.0353775.ref027","doi-asserted-by":"crossref","first-page":"2305","DOI":"10.1126\/science.286.5448.2305","article-title":"Structure of a Transcribing T7 RNA Polymerase Initiation Complex","volume":"286","author":"GMT Cheetham","year":"1999","journal-title":"Science"},{"issue":"5597","key":"pone.0353775.ref028","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1126\/science.1077464","article-title":"Structural Basis for the Transition from Initiation to Elongation Transcription in T7 RNA Polymerase","volume":"298","author":"YW Yin","year":"2002","journal-title":"Science"},{"issue":"6911","key":"pone.0353775.ref029","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1038\/nature01129","article-title":"Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution","volume":"420","author":"TH Tahirov","year":"2002","journal-title":"Nature"},{"key":"pone.0353775.ref030","author":"Promega Corporation","year":"2016"},{"issue":"34","key":"pone.0353775.ref031","doi-asserted-by":"crossref","first-page":"24441","DOI":"10.1074\/jbc.M604369200","article-title":"Observed Instability of T7 RNA Polymerase Elongation Complexes Can Be Dominated by Collision-induced \u201cBumping\u201d","volume":"281","author":"Y Zhou","year":"2006","journal-title":"J Biol Chem"},{"issue":"46","key":"pone.0353775.ref032","doi-asserted-by":"crossref","first-page":"19838","DOI":"10.1073\/pnas.1012985107","article-title":"Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles","volume":"107","author":"BD Allen","year":"2010","journal-title":"Proc Natl Acad Sci USA"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0353775","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T17:28:51Z","timestamp":1784568531000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0353775"}},"subtitle":[],"editor":[{"given":"Jian","family":"Xu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,7,20]]},"references-count":32,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7,20]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0353775","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,20]]}}}