{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T17:44:27Z","timestamp":1792691067173,"version":"4.5.0"},"reference-count":27,"publisher":"Public Library of Science (PLoS)","issue":"10","license":[{"start":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T00:00:00Z","timestamp":1792627200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.<\/jats:p>","DOI":"10.1371\/journal.pone.0354008","type":"journal-article","created":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T17:35:06Z","timestamp":1792690506000},"page":"e0354008","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["High-throughput thermodynamic fingerprinting of protein\u2013ligand interactions by DNA-directed focal molography"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-0489-4588","authenticated-orcid":true,"given":"John","family":"Oehninger","sequence":"first","affiliation":[{"name":"lino Biotech AG","place":["Zurich, Switzerland"],"department":["R\u2009&\u2009D"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simona","family":"Notova","sequence":"additional","affiliation":[{"name":"lino Biotech AG","place":["Zurich, Switzerland"],"department":["R\u2009&\u2009D"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2523-8918","authenticated-orcid":true,"given":"Andreas","family":"Frutiger","sequence":"additional","affiliation":[{"name":"lino Biotech AG","place":["Zurich, Switzerland"],"department":["R\u2009&\u2009D"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,10,22]]},"reference":[{"issue":"10","key":"pone.0354008.ref001","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1038\/nrd.2016.123","article-title":"Biophysics in drug discovery: impact, challenges and opportunities","volume":"15","author":"J-P Renaud","year":"2016","journal-title":"Nat Rev Drug Discov"},{"key":"pone.0354008.ref002","doi-asserted-by":"crossref","DOI":"10.1039\/9781788010283","volume-title":"Handbook of Surface Plasmon Resonance","author":"RBM Schasfoort","year":"2017","edition":"2"},{"issue":"2","key":"pone.0354008.ref003","article-title":"Quantitative analysis of ligand-EGFR interactions: a platform for screening targeting molecules","volume":"10","author":"W-T Kuo","year":"2015","journal-title":"PLoS One"},{"issue":"12","key":"pone.0354008.ref004","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pcbi.1006601","article-title":"Determinants of drug-target interactions at the single cell level","volume":"14","author":"V Elgart","year":"2018","journal-title":"PLoS Comput Biol"},{"issue":"11","key":"pone.0354008.ref005","doi-asserted-by":"crossref","first-page":"970","DOI":"10.1016\/j.tibs.2016.08.013","article-title":"Macromolecular crowding in vitro, in vivo, and in between","volume":"41","author":"G Rivas","year":"2016","journal-title":"Trends in Biochemical Sciences"},{"issue":"1","key":"pone.0354008.ref006","doi-asserted-by":"crossref","first-page":"14169","DOI":"10.1038\/s41598-017-14257-4","article-title":"From receptor binding kinetics to signal transduction; 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