{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T13:23:19Z","timestamp":1787404999416,"version":"build-2736575974"},"reference-count":46,"publisher":"American Chemical Society (ACS)","issue":"8","license":[{"start":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T00:00:00Z","timestamp":1785110400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000165","name":"Division of Chemistry","doi-asserted-by":"publisher","award":["CHE-1752924"],"award-info":[{"award-number":["CHE-1752924"]}],"id":[{"id":"10.13039\/100000165","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001309","name":"Research Corporation for Science Advancement","doi-asserted-by":"publisher","award":["CS-SEED-2004-006"],"award-info":[{"award-number":["CS-SEED-2004-006"]}],"id":[{"id":"10.13039\/100001309","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001309","name":"Research Corporation for Science Advancement","doi-asserted-by":"publisher","award":["CS-SEED-2024-006"],"award-info":[{"award-number":["CS-SEED-2024-006"]}],"id":[{"id":"10.13039\/100001309","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,21]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Xeno Nucleic Acids (XNA) are chemically modified versions of DNA. These modifications can bypass natural immune responses and impart novel properties in complex biological systems. Previous efforts have identified mutant proteins capable of robust XNA synthesis; one mutant, SFM4-3, has been shown to be able to amplify partially substituted XNA in the polymerase chain reaction (PCR) which is necessary for many applications of XNA. However, SFM4-3 mediated PCR is inefficient and has required up to 22 h of reaction time to create amplicons of \u223c75 nucleotides (nt); optimizing the PCR amplification of XNA can potentially improve its utility. Here, we show an optimized method for XNA PCR and compare the XNA PCR ability of mutant XNA polymerase enzymes. We show that SFP1 and SFP4 perform better than SFM4-3 in the amplification of 2\u2032F-XNA. We also highlight the role of protein stability in XNA polymerase PCR performance, demonstrating that significantly diminished thermostability may limit current XNA polymerases. This work decreases the time needed for XNA PCR to &amp;lt;4 h, increases the length of amplicons generated to 245 nt, identifies new PCR-active enzymes, and provides guidance for how to improve XNA polymerases for PCR in the future.<\/jats:p>","DOI":"10.1021\/acschembio.6c00270","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T13:10:01Z","timestamp":1785849001000},"page":"1927-1936","source":"Crossref","is-referenced-by-count":0,"title":["Efforts Toward XNA PCR: Optimization and Characterization\nof PCR-Amplification of Chimeric 2\u2032F XNA\/DNA"],"prefix":"10.1021","volume":"21","author":[{"given":"Helen V.","family":"Branson","sequence":"first","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aria A.","family":"Khalique","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mia J.","family":"Kronson","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Delaney I.","family":"Carlin","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Avery E.","family":"Roof","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madison K.C.","family":"Seto","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ysabel","family":"David","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Cao","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susanna E.","family":"Barrett","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taiasean","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5789-9147","authenticated-orcid":true,"given":"Aaron M.","family":"Leconte","sequence":"additional","affiliation":[{"name":"Department of Natural Sciences of Pitzer and Scripps College , , ,","place":["Claremont, California, United States of America, 91711"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"issue":"63","key":"2026082208552632000_cit1","doi-asserted-by":"publisher","DOI":"10.3791\/3998","article-title":"Polymerase\nchain reaction: basic protocol plus troubleshooting and optimization\nstrategies","author":"Lorenz","year":"2012","journal-title":"J. Vis. Exp."},{"issue":"6","key":"2026082208552632000_cit2","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1016\/S1473-3099(04)01044-8","article-title":"PCR-based diagnostics for infectious diseases: uses,\nlimitations, and future applications in acute-care settings","volume":"4","author":"Yang","year":"2004","journal-title":"Lancet Infect. Dis."},{"key":"2026082208552632000_cit3","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1002\/ddr.10378","article-title":"Quantitative\nreal time polymerase chain reaction in drug development","volume":"62","author":"Goodsaid","year":"2004","journal-title":"Drug Dev Res"},{"issue":"7","key":"2026082208552632000_cit4","doi-asserted-by":"publisher","first-page":"3976","DOI":"10.1021\/acs.chemrev.3c00028","article-title":"Recent\nAdvances in DNA Origami-Engineered Nanomaterials\nand Applications","volume":"123","author":"Zhan","year":"2023","journal-title":"Chem. Rev."},{"issue":"23","key":"2026082208552632000_cit5","doi-asserted-by":"publisher","first-page":"4229","DOI":"10.3390\/molecules24234229","article-title":"A Review of Their\nChemical Properties and Modifications\nfor Therapeutic Application","volume":"24","author":"Adachi","year":"2019","journal-title":"Molecules"},{"issue":"7","key":"2026082208552632000_cit6","doi-asserted-by":"publisher","first-page":"595","DOI":"10.1016\/j.tibs.2016.04.010","article-title":"Catalytic\nDNA: Scope, Applications, and Biochemistry of Deoxyribozymes","volume":"41","author":"Silverman","year":"2016","journal-title":"Trends Biochem. Sci."},{"issue":"6","key":"2026082208552632000_cit7","doi-asserted-by":"publisher","first-page":"556","DOI":"10.1038\/nchem.2493","article-title":"Evolution of thermophilic DNA polymerases\nfor the recognition\nand amplification of C2\u2019-modified DNA","volume":"8","author":"Chen","year":"2016","journal-title":"Nat. Chem."},{"issue":"12","key":"2026082208552632000_cit8","doi-asserted-by":"publisher","first-page":"8179","DOI":"10.1039\/C7SC03747C","article-title":"Evolved\npolymerases facilitate selection\nof fully 2\u2019-OMe-modified aptamers","volume":"8","author":"Liu","year":"2017","journal-title":"Chem.\nSci."},{"issue":"34","key":"2026082208552632000_cit9","doi-asserted-by":"publisher","first-page":"11570","DOI":"10.1002\/anie.201905999","article-title":"What Is XNA?","volume":"58","author":"Chaput","year":"2019","journal-title":"Angew. Chem. Int.\nEd."},{"issue":"11","key":"2026082208552632000_cit10","doi-asserted-by":"publisher","first-page":"1667","DOI":"10.1261\/rna.077008.120","article-title":"2\u2019-fluoro-modified\npyrimidines enhance affinity of RNA oligonucleotides to HIV-1 reverse\ntranscriptase","volume":"26","author":"Gruenke","year":"2020","journal-title":"RNA"},{"issue":"21","key":"2026082208552632000_cit11","doi-asserted-by":"publisher","first-page":"6690","DOI":"10.1021\/jacs.8b03447","article-title":"Phosphonomethyl Oligonucleotides\nas Backbone-Modified Artificial Genetic Polymers","volume":"140","author":"Liu","year":"2018","journal-title":"J. Am. Chem. Soc."},{"issue":"9","key":"2026082208552632000_cit12","doi-asserted-by":"publisher","first-page":"1616","DOI":"10.1002\/cbdv.201100081","article-title":"An overview\nof sugar-modified oligonucleotides for antisense therapeutics","volume":"8","author":"Prakash","year":"2011","journal-title":"Chem. 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Commun."},{"issue":"47","key":"2026082208552632000_cit16","doi-asserted-by":"publisher","first-page":"25789","DOI":"10.1021\/jacs.3c09497","article-title":"Functionally\nEnhanced XNA Aptamers Discovered by Parallelized\nLibrary Screening","volume":"145","author":"Lozoya-Colinas","year":"2023","journal-title":"J. Am. Chem. 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Biol."},{"issue":"8","key":"2026082208552632000_cit25","doi-asserted-by":"publisher","first-page":"2892","DOI":"10.1021\/jacs.6b13132","article-title":"Selection of 2\u2019-Fluoro-Modified\nAptamers with\nOptimized Properties","volume":"139","author":"Thirunavukarasu","year":"2017","journal-title":"J. Am. Chem. Soc."},{"issue":"5","key":"2026082208552632000_cit26","doi-asserted-by":"publisher","first-page":"2125","DOI":"10.1021\/jacs.9b10538","article-title":"Selection of Aptamers with Large\nHydrophobic 2\u2019-Substituents","volume":"142","author":"Shao","year":"2020","journal-title":"J. Am. Chem.\nSoc."},{"issue":"45","key":"2026082208552632000_cit27","doi-asserted-by":"publisher","first-page":"14046","DOI":"10.1002\/anie.201707367","article-title":"Enzymatic Synthesis, Amplification, and Application\nof DNA with a Functionalized Backbone","volume":"56","author":"Chen","year":"2017","journal-title":"Angew.\nChem. Int. Ed."},{"issue":"6","key":"2026082208552632000_cit28","doi-asserted-by":"publisher","first-page":"1748","DOI":"10.1021\/ja038525p","article-title":"Expanding\nthe substrate repertoire of a DNA polymerase by directed evolution","volume":"126","author":"Fa","year":"2004","journal-title":"J. Am. Chem. Soc."},{"issue":"38","key":"2026082208552632000_cit29","doi-asserted-by":"publisher","first-page":"5999","DOI":"10.1021\/acs.biochem.5b00689","article-title":"Taq DNA Polymerase Mutants and 2\u2019-Modified\nSugar Recognition","volume":"54","author":"Schultz","year":"2015","journal-title":"Biochemistry"},{"key":"2026082208552632000_cit30","doi-asserted-by":"publisher","DOI":"10.1021\/acsomega.5c07370","article-title":"Enzymatic\nSynthesis of Mixed XNA Polymers Containing\n2\u2032Fluoro and 2\u2032Azide Modifications","author":"Kang","year":"2025","journal-title":"ACS Omega"},{"issue":"8","key":"2026082208552632000_cit31","doi-asserted-by":"publisher","first-page":"1044","DOI":"10.1039\/D2CB00064D","article-title":"Mutant\npolymerases capable of 2\u2019 fluoro-modified nucleic acid synthesis\nand amplification with improved accuracy","volume":"3","author":"Christensen","year":"2022","journal-title":"RSC\nChem. Biol."},{"issue":"21","key":"2026082208552632000_cit32","doi-asserted-by":"publisher","first-page":"11982","DOI":"10.1093\/nar\/gkaa999","article-title":"Enzymatic\nsynthesis of hypermodified\nDNA polymers for sequence-specific display of four different hydrophobic\ngroups","volume":"48","author":"Ondrus\u030c","year":"2020","journal-title":"Nucleic Acids Res."},{"issue":"1","key":"2026082208552632000_cit33","doi-asserted-by":"publisher","first-page":"9897","DOI":"10.1038\/s41467-025-65808-7","article-title":"Beyond the niche -\nunlocking the\nfull potential of synthetic riboswitches","volume":"16","author":"Hoetzel","year":"2025","journal-title":"Nat.\nCommun."},{"issue":"18","key":"2026082208552632000_cit34","doi-asserted-by":"publisher","first-page":"11616","DOI":"10.1073\/pnas.182095699","article-title":"Isolation of high-affinity\nGTP aptamers from partially\nstructured RNA libraries","volume":"99","author":"Davis","year":"2002","journal-title":"Proc. Natl. Acad.\nSci. U. S. 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Ed."},{"issue":"6","key":"2026082208552632000_cit38","doi-asserted-by":"publisher","first-page":"755","DOI":"10.1038\/nbt974","article-title":"Generic expansion\nof the substrate spectrum of a DNA polymerase by directed evolution","volume":"22","author":"Ghadessy","year":"2004","journal-title":"Nat. Biotechnol."},{"issue":"17","key":"2026082208552632000_cit39","doi-asserted-by":"publisher","first-page":"3115","DOI":"10.1002\/anie.200603987","article-title":"Evolving a thermostable DNA polymerase\nthat amplifies from highly damaged templates","volume":"46","author":"Gloeckner","year":"2007","journal-title":"Angew. Chem. Int. Ed."},{"issue":"8","key":"2026082208552632000_cit40","doi-asserted-by":"publisher","first-page":"4552","DOI":"10.1073\/pnas.071052198","article-title":"Directed evolution of polymerase\nfunction by compartmentalized self-replication","volume":"98","author":"Ghadessy","year":"2001","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"21","key":"2026082208552632000_cit41","doi-asserted-by":"publisher","first-page":"6507","DOI":"10.1007\/s00253-023-12744-5","article-title":"Customized multiple\nsequence alignment as an effective strategy to improve performance\nof Taq DNA polymerase","volume":"107","author":"Li","year":"2023","journal-title":"Appl. Microbiol. Biotechnol."},{"issue":"15","key":"2026082208552632000_cit42","doi-asserted-by":"publisher","first-page":"5869","DOI":"10.1073\/pnas.0510098103","article-title":"Protein stability\npromotes evolvability","volume":"103","author":"Bloom","year":"2006","journal-title":"Proc. Natl. Acad. Sci.\nU. S. A."},{"issue":"3","key":"2026082208552632000_cit43","doi-asserted-by":"publisher","first-page":"785","DOI":"10.1042\/bj20030323","article-title":"Comparative thermal\ndenaturation of Thermus aquaticus and Escherichia coli type 1 DNA\npolymerases","volume":"374","author":"Karantzeni","year":"2003","journal-title":"Biochem. J."},{"issue":"1","key":"2026082208552632000_cit44","doi-asserted-by":"publisher","first-page":"925","DOI":"10.1038\/s41467-025-67652-1","article-title":"Directed evolution of a TNA polymerase identifies\nindependent paths to fidelity and catalysis","volume":"17","author":"Hajjar","year":"2025","journal-title":"Nat. Commun."},{"issue":"6079","key":"2026082208552632000_cit45","doi-asserted-by":"publisher","first-page":"341","DOI":"10.1126\/science.1217622","article-title":"Synthetic genetic polymers capable of heredity\nand evolution","volume":"336","author":"Pinheiro","year":"2012","journal-title":"Science"},{"key":"2026082208552632000_cit46","doi-asserted-by":"publisher","first-page":"960","DOI":"10.1038\/s41589-025-02124-7","article-title":"Rapid evolution\nof a highly efficient RNA polymerase by homologous recombination","volume":"22","author":"Medina","year":"2026","journal-title":"Nat. Chem. Biol."}],"container-title":["ACS Chemical Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acschembio.6c00270","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/21\/8\/1927\/66286869\/acschembio.6c00270.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/21\/8\/1927\/66286869\/acschembio.6c00270.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T12:55:43Z","timestamp":1787403343000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acbcct\/article\/21\/8\/1927\/5234357\/Efforts-Toward-XNA-PCR-Optimization-and"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,27]]},"references-count":46,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,7,27]]},"published-print":{"date-parts":[[2026,8,21]]}},"URL":"https:\/\/doi.org\/10.1021\/acschembio.6c00270","relation":{},"ISSN":["1554-8929","1554-8937"],"issn-type":[{"value":"1554-8929","type":"print"},{"value":"1554-8937","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,27]]}}}