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In this study, we focused on yellow fever virus (YFV) as a model system to design and validate a generalizable de novo protein design framework integrating evolutionary genomics with structural modeling to rationally engineer diagnostic antigens with reduced cross-reactivity and improved cost-effectiveness. Through comprehensive evolutionary mapping over time of the YFV polyprotein, we identified conserved yet discriminatory peptide regions within the NS1 protein and selected one peptide to guide scaffold-based protein design while preserving critical structural constraints. As a complementary strategy to develop antibody alternatives for antigen detection assays, we also implemented an in silico RNA aptamer design pipeline to circumvent the time and cost limitations of conventional in vitro Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique. Nevertheless, NS1-targeting aptamers demonstrated limited sensitivity and specificity upon experimental validation. In contrast, structure-guided design of the NS1-derived protein (YFV-Scaffold) yielded a thermally stable, highly expressible recombinant protein, which demonstrated sensitivity and specificity comparable to those of commercially available full-length NS1 in in-house ELISA assays. Circular dichroism analysis revealed minimal denaturation up to 94 \u00b0C and complete recovery of secondary structure upon cooling, underscoring its remarkable structural resilience. Collectively, our results establish an evolutionary dynamics-aware strategy for target-antigen design, providing a scalable platform for the rational design of robust, low-cost diagnostic proteins applicable to a wide range of viral pathogens.<\/jats:p>","DOI":"10.1021\/acsomega.6c07158","type":"journal-article","created":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T10:01:55Z","timestamp":1788516115000},"page":"56323-56335","source":"Crossref","is-referenced-by-count":0,"title":["Evolutionary Dynamics-Aware\n                    <i>De Novo<\/i>\n                    Design of a Thermostable NS1-Derived Diagnostic\nProtein for Yellow\nFever Virus"],"prefix":"10.1021","volume":"11","author":[{"given":"Junior Olimpio","family":"Martins","sequence":"first","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 04023-062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patri\u0301cia da Silva","family":"Antunes","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 04023-062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rodrigo Lopes Sanz","family":"Duro","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 04023-062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4506-3756","authenticated-orcid":true,"given":"Robert","family":"Andreata-Santos","sequence":"additional","affiliation":[{"name":"Institut Pasteur de Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 05508-020"]},{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 04023-062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clara Lacerda de","family":"Athayde","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 04023-062"]},{"name":"Anhembi Morumbi University , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 04546-001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gustavo","family":"Cabral-Miranda","sequence":"additional","affiliation":[{"name":"University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 05403-000"]},{"name":"University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 05403-000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-1824-7492","authenticated-orcid":true,"given":"Luiz Ma\u0301rio","family":"Ramos Janini","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 04023-062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Franca","family":"Fraternali","sequence":"additional","affiliation":[{"name":"University College London , , Darwin Building, 604 Gower Street , ,","place":["London, U.K., WC1E 6BT"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos Henrique Bezerra da","family":"Cruz","sequence":"additional","affiliation":[{"name":"University College London , , Darwin Building, 604 Gower Street , ,","place":["London, U.K., WC1E 6BT"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5734-7601","authenticated-orcid":true,"given":"Ricardo","family":"Dura\u0303es-Carvalho","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 04023-062"]},{"name":"University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, Sa\u0303o Paulo, Brazil, 05508-000"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,4]]},"reference":[{"issue":"3","key":"2026092211022078800_cit1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0007213","article-title":"Global Expansion\nand Redistribution of Aedes-Borne Virus Transmission Risk with Climate\nChange","volume":"13","author":"Ryan","year":"2019","journal-title":"PLoS Neglected Trop. 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