{"title":"How reliable is the evaluation of DNA binding constants? Insights and best practices based on an inter-laboratory fluorescence titration study","authors":"","doi":"10.1016/j.saa.2024.125354","DOIUrl":null,"url":null,"abstract":"<div><div>In 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 (log<em>K</em>) values not falling below 5 % relative standard deviation (RSD), or ± 0.5 log<em>K</em> 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.</div></div>","PeriodicalId":433,"journal":{"name":"Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy","FirstCategoryId":"92","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1386142524015208","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"SPECTROSCOPY","Score":null,"Total":0}
引用次数: 0
Abstract
In 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.
期刊介绍:
Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy (SAA) is an interdisciplinary journal which spans from basic to applied aspects of optical spectroscopy in chemistry, medicine, biology, and materials science.
The journal publishes original scientific papers that feature high-quality spectroscopic data and analysis. From the broad range of optical spectroscopies, the emphasis is on electronic, vibrational or rotational spectra of molecules, rather than on spectroscopy based on magnetic moments.
Criteria for publication in SAA are novelty, uniqueness, and outstanding quality. Routine applications of spectroscopic techniques and computational methods are not appropriate.
Topics of particular interest of Spectrochimica Acta Part A include, but are not limited to:
Spectroscopy and dynamics of bioanalytical, biomedical, environmental, and atmospheric sciences,
Novel experimental techniques or instrumentation for molecular spectroscopy,
Novel theoretical and computational methods,
Novel applications in photochemistry and photobiology,
Novel interpretational approaches as well as advances in data analysis based on electronic or vibrational spectroscopy.