{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:39:03Z","timestamp":1760060343922,"version":"build-2065373602"},"reference-count":32,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T00:00:00Z","timestamp":1755561600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Portuguese Foundation for Science and Technology (FCT)","award":["UIDB\/04565\/2020","UIDP\/04565\/2020","LA\/P\/0140\/2020"],"award-info":[{"award-number":["UIDB\/04565\/2020","UIDP\/04565\/2020","LA\/P\/0140\/2020"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Plasmid DNA (pDNA) purification plays a key role in the development of vaccines and gene therapies. Affinity chromatography stands out as a promising method for plasmid purification, leveraging a range of biological and synthetic ligands to achieve selectivity. This study investigates the potential of a synthetic ligand library consisting of triazine-based bifunctional compounds designed to mimic the side chains of amino acids that are known to bind nucleic acids. A high-throughput screening method was employed to assess the binding ability of 158 ligands within the library to single-stranded, FITC-labeled homo-oligonucleotides (G and T), each comprising 20 nucleotides, under both hydrophilic and hydrophobic conditions. High-affinity ligands were identified for both T and G oligonucleotides. Follow-up microscale chromatographic screening uncovered some false positives from the initial FITC-based screening, narrowing the selection to 22 ligands for further investigation. In the next phase of the study, the binding affinity of these ligands towards double-stranded oligonucleotides (AT and CG) was assessed. Ligand 1\/2, a mimic of Ala-Lys or Gly-Lys, and ligand 2\/3, a mimic of Lys-Tyr, were chosen as initial candidates for evaluating plasmid DNA purification from an Escherichia coli crude extract. The results obtained with 0.4 M ammonium sulfate in 20 mM Tris-HCl (pH 8.0) as the binding buffer were similar to those observed when purifying plasmid DNA from E. coli clarified lysates by hydrophobic interaction chromatography. The affinity resins retained RNA, while the less hydrophobic plasmid DNA was excluded in the initial fractions. Future research will be directed towards exploring the potential of the most promising ligands to separate pDNA isoforms.<\/jats:p>","DOI":"10.3390\/molecules30163423","type":"journal-article","created":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T15:29:29Z","timestamp":1755617369000},"page":"3423","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Screening of a Combinatorial Library of Triazine-Scaffolded Dipeptide-Mimic Affinity Ligands to Bind Plasmid DNA"],"prefix":"10.3390","volume":"30","author":[{"given":"Jo\u00e3o F. R.","family":"Belchior","sequence":"first","affiliation":[{"name":"iBB\u2014Institute for Bioengineering and Biosciences, and Associate Laboratory i4HB\u2014Institute for Health and Bioeconomy at Instituto Superior T\u00e9cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal"},{"name":"Bioengineering Department, Instituto Superior T\u00e9cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4840-6299","authenticated-orcid":false,"given":"Gabriel A.","family":"Monteiro","sequence":"additional","affiliation":[{"name":"iBB\u2014Institute for Bioengineering and Biosciences, and Associate Laboratory i4HB\u2014Institute for Health and Bioeconomy at Instituto Superior T\u00e9cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal"},{"name":"Bioengineering Department, Instituto Superior T\u00e9cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6928-692X","authenticated-orcid":false,"given":"D. Miguel","family":"Prazeres","sequence":"additional","affiliation":[{"name":"iBB\u2014Institute for Bioengineering and Biosciences, and Associate Laboratory i4HB\u2014Institute for Health and Bioeconomy at Instituto Superior T\u00e9cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal"},{"name":"Bioengineering Department, Instituto Superior T\u00e9cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3776-180X","authenticated-orcid":false,"given":"M. \u00c2ngela","family":"Taipa","sequence":"additional","affiliation":[{"name":"iBB\u2014Institute for Bioengineering and Biosciences, and Associate Laboratory i4HB\u2014Institute for Health and Bioeconomy at Instituto Superior T\u00e9cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal"},{"name":"Bioengineering Department, Instituto Superior T\u00e9cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1186\/s12985-025-02645-6","article-title":"Revolutionizing immunization: A comprehensive review of mRNA vaccine technology and applications","volume":"22","author":"Leong","year":"2025","journal-title":"Virol. J."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1089\/hum.2017.159","article-title":"Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications","volume":"28","author":"Schmeer","year":"2017","journal-title":"Hum. Gene Ther."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1891","DOI":"10.1016\/j.drudis.2020.09.040","article-title":"Plasmid manufacture is the bottleneck of the genetic medicine revolution","volume":"25","author":"Ohlson","year":"2020","journal-title":"Drug Discov. 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