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

[Display omitted] •Ethidium-DNA is chosen as a model for a study extendable to other systems/techniques.•Fluorescence titration best practices are discussed and a protocol is proposed.•Data fitting pros and cons using different equations and software are discussed.•Binding constant and DNA site dimension dispersions are analysed.•An unavoidable uncertainty of 5% (log K rsd) is evidenced. 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.