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How reliable is the evaluation of DNA binding constants? insights and best practices based on an inter-laboratory fluorescence titration study

2025-02Artigo científico Acesso aberto
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datacite.subject.sdg09:Indústria, Inovação e Infraestruturas
datacite.subject.sdg03:Saúde de Qualidade
datacite.subject.sdg04:Educação de Qualidade
dc.contributor.authorDömötör, Orsolya
dc.contributor.authorBinacchi, Francesca
dc.contributor.authorRibeiro, Nádia
dc.contributor.authorBusto, Natalia
dc.contributor.authorGonzalez-García, Jorge
dc.contributor.authorGarcia-España, Enrique
dc.contributor.authorCorreia, Isabel
dc.contributor.authorEnyedy, Éva A.
dc.contributor.authorHamacek, Josef
dc.contributor.authorTerenzi, Alessio
dc.contributor.authorBasílio, Nuno
dc.contributor.authorBarone, Giampaolo
dc.contributor.authorPALMA ANTUNES CAVACO, ISABEL MARIA
dc.contributor.authorBiver, Tarita
dc.date.accessioned2026-04-30T12:37:59Z
dc.date.available2026-04-30T12:37:59Z
dc.date.issued2025-02
dc.description.abstractIn all experimental sciences, the precision and reliability of quantitative measurements are paramount. This is particularly true when examining the interactions between small molecules and biomolecules/polyelectrolytes, such as DNAs/RNAs, and yet it is overlooked in most publications of thermodynamic binding parameters. This paper presents findings from COST Action 18202 “Network for Equilibria and Chemical Thermodynamics Advanced Research,” which assessed the consistency of data derived from the interactions of calf-thymus DNA (CT-DNA) with the fluorescent intercalator ethidium bromide (EB) through spectrofluorimetric titrations. We first discuss critical experimental aspects and propose a reference experimental protocol which can be used to calibrate procedures for the determination of nucleic acid binding equilibrium constants. We then fit the experimental points according to different procedures and analyse the results focusing on the statistical dispersion of the data, aiming at enlightening the strong and weak points of different fitting procedures. The implications of this work are significant, demonstrating how the statistical dispersion of experimental data can influence the interpretation of biochemical coordination mechanisms. Our study reveals that, despite rigorous protocol standardization, the determination of binding parameters remains sensitive to the choice of data fitting method, with deviations in the logarithmic stability constant (logK) values not falling below 5 % relative standard deviation (RSD), or ± 0.5 logK units for 95 % confidence. This variability evidences the critical need for standardized best practices in data treatment as well as experimental procedures. Although our study focuses on the EB/CT-DNA system through fluorescence titrations, the broader implications for other methodologies across various biochemical systems highlight the importance of this first-of-its-kind inter-laboratory comparison in advancing our understanding of biochemical coordination processes.eng
dc.description.sponsorshipLA/P/0008/2020; CEECIND/00466/2017/CP1462/CT0013
dc.identifier.doi10.1016/j.saa.2024.125354
dc.identifier.issn1386-1425
dc.identifier.urihttp://hdl.handle.net/10400.1/28828
dc.language.isoeng
dc.peerreviewedyes
dc.publisherElsevier
dc.relationCentro de Química Estrutural
dc.relationCentro de Química Estrutural
dc.relationInstitute of Molecular Sciences
dc.relationAssociated Laboratory for Green Chemistry - Clean Technologies and Processes
dc.relationAssociated Laboratory for Green Chemistry - Clean Technologies and Processes
dc.relation.ispartofSpectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectSite size
dc.subjectSoftware
dc.subjectData fitting
dc.titleHow reliable is the evaluation of DNA binding constants? insights and best practices based on an inter-laboratory fluorescence titration studyeng
dc.typejournal article
dspace.entity.typePublication
oaire.awardNumberUIDB/00100/2020
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oaire.awardNumberUIDB/50006/2020
oaire.awardTitleCentro de Química Estrutural
oaire.awardTitleCentro de Química Estrutural
oaire.awardTitleInstitute of Molecular Sciences
oaire.awardTitleAssociated Laboratory for Green Chemistry - Clean Technologies and Processes
oaire.awardTitleAssociated Laboratory for Green Chemistry - Clean Technologies and Processes
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F00100%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F00100%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/LA%2FP%2F0056%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F50006%2F2020/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F50006%2F2020/PT
oaire.citation.startPage125354
oaire.citation.titleSpectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
oaire.citation.volume327
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream6817 - DCRRNI ID
oaire.versionhttp://purl.org/coar/version/c_970fb48d4fbd8a85
person.familyNamePALMA ANTUNES CAVACO
person.givenNameISABEL MARIA
person.identifierH-9087-2012
person.identifier.ciencia-idD515-FB5E-1205
person.identifier.orcid0000-0001-8651-9054
project.funder.identifierhttp://doi.org/10.13039/501100001871
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