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How reliable is the evaluation of DNA binding constants?

2025-02-15Artigo científico Acesso aberto
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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.authorCavaco, Isabel
dc.contributor.authorBiver, Tarita
dc.contributor.institutionLAQV@REQUIMTE
dc.contributor.institutionDQ - Departamento de Química
dc.contributor.pblElsevier Science Publisher B.V.
dc.date.accessioned2025-07-01T21:15:47Z
dc.date.available2025-07-01T21:15:47Z
dc.date.issued2025-02-15
dc.descriptionFunding Information: This contribution is based upon work from COST Action CA18202, NECTAR—Network for Equilibria and Chemical Thermodynamics Advanced Research, supported by COST (European Cooperation in Science and Technology). This work was also supported by the National Research, Development and Innovation Office-NKFIA (Hungary) through project TKP-2021-EGA-32, Fundação para a Ciência e Tecnologia (Portugal) through projects UIDB/00100/2020 (DOI: 10.54499/UIDB/00100/2020), UIDP/00100/2020 (DOI: 10.54499/UIDP/00100/2020) and LA/P/0056/2020 (DOI: 10.54499/LA/P/0056/2020), LA/P/0008/2020 (DOI: 10.54499/LA/P/0008/2020), UIDP/50006/2020 (DOI: 10.54499/UIDP/50006/2020), UIDB/50006/2020 (DOI: 10.54499/UIDB/50006/2020), CEECIND/00466/2017/CP1462/CT0013 (DOI: 10.54499/CEECIND/00466/2017/CP1462/CT0013), Consellería de Universidades, Ciencia y Sociedad Digital of the Generalitat Valenciana (PROMETEO Grant CIPROM/2021/030). Publisher Copyright: © 2024 The Author(s)
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.en
dc.description.versionpublishersversion
dc.description.versionpublished
dc.format.extent12
dc.format.extent1791852
dc.identifier.doi10.1016/j.saa.2024.125354
dc.identifier.issn1386-1425
dc.identifier.otherPURE: 104743214
dc.identifier.otherPURE UUID: 82f4e8b6-5b8b-4215-956f-2b8e476a635a
dc.identifier.otherScopus: 85208055259
dc.identifier.otherWOS: 001350894300001
dc.identifier.otherPubMed: 39500201
dc.identifier.urihttp://hdl.handle.net/10362/184698
dc.identifier.urlhttps://www.scopus.com/pages/publications/85208055259
dc.identifier.urlhttps://www.webofscience.com/wos/woscc/full-record/WOS:001350894300001
dc.language.isoeng
dc.peerreviewedyes
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dc.relationinfo:eu-repo/grantAgreement/FCT/CEEC IND 2017/CEECIND%2F00466%2F2017%2FCP1462%2FCT0013/PT
dc.relationNot Available
dc.subjectData fitting
dc.subjectMulti-wavelength
dc.subjectScatchard plot
dc.subjectSite size
dc.subjectSoftware
dc.subjectAnalytical Chemistry
dc.subjectAtomic and Molecular Physics, and Optics
dc.subjectInstrumentation
dc.subjectSpectroscopy
dc.titleHow reliable is the evaluation of DNA binding constants?en
dc.title.subtitleInsights and best practices based on an inter-laboratory fluorescence titration studyen
dc.typejournal article
degois.publication.firstPage1
degois.publication.lastPage12
degois.publication.titleSpectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
degois.publication.volume327
dspace.entity.typePublication
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oaire.awardURIinfo:eu-repo/grantAgreement/FCT/CEEC IND 2017/CEECIND%2F00466%2F2017%2FCP1462%2FCT0013/PT
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oaire.fundingStreamConcurso de avaliação no âmbito do Programa Plurianual de Financiamento de Unidades de I&D (2017/2018) - Financiamento Programático
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