[Paper Review] Calculating the contribution of different binding modes to Quinacrine - DNA complex formation from polarized fluorescence data

Igor Voloshin, Olga Ryazanova+2 more|arXiv (Cornell University)|Nov 25, 2013

DNA and Nucleic Acid Chemistry1 references3 citations

TL;DR

This study uses polarized fluorescence spectroscopy to quantify the contributions of intercalative and electrostatic binding modes of quinacrine (QA) to chicken erythrocyte DNA across varying phosphate-to-dye (P/D) ratios and ionic strengths. It reveals that electrostatic binding, though weaker, dominates at low P/D due to a smaller binding site size (8× smaller than intercalation), and presents a method to deconvolute binding mode contributions from fluorescence titration data with quantitative parameterization of complex formation.

ABSTRACT

Binding of acridine derivative quinacrine (QA) to chicken erythrocyte DNA was studied by methods of absorption and polarized fluorescent spectroscopy. Measurements were carried out in aqueous buffered solutions (pH 6.9) of different dye concentrations (QA concentration range from $10^{-6}$ till $10^{-4}$ M) and ionic strengths ($Na^{+}$ concentration rang from $10^{-3}$ till 0.15 M) in a wide range of phosphate-to-dye molar ratios ($P/D$). It is established that the minimum of fluorescent titration curve plotted as relative fluorescence intensity $vs$ $P/D$ is conditioned by the competition between the two types of QA binding to DNA which posses by different emission parameters: (i) intercalative one dominating under high $P/D$ values, and (ii) outside electrostatic binding dominating under low $P/D$ values, which is accompanied by the formation of non-fluorescent dye associates on the DNA backbone. Absorption and fluorescent characteristics of complexes formed were determined. The method of calculation of different binding modes contribution to the complex formation depending on $P/D$ value is presented. It was shown that the size of binding site measured as the number of DNA base pairs per one QA molecule bound in the case of the electrostatic interaction is 8 times less than that for the intercalative one that determines the competitive ability of the outside binding against the stronger intercalative binding mode.

Motivation & Objective

• To determine the relative contributions of intercalative and electrostatic binding modes of quinacrine (QA) to DNA under varying ionic strength and P/D ratios.
• To resolve the competition between two distinct QA-DNA binding modes using polarized fluorescence spectroscopy.
• To develop a quantitative method for calculating the fraction of each binding mode in the complex formation process.
• To measure and compare the binding site sizes for intercalative and electrostatic binding modes.
• To correlate changes in fluorescence intensity and polarization with the formation of non-fluorescent dye associates during electrostatic binding.

Proposed method

• Polarized fluorescence spectroscopy was used to measure relative fluorescence intensity and anisotropy as a function of P/D molar ratio and ionic strength.
• Absorption and fluorescence titration curves were recorded across QA concentrations from 10⁻⁶ M to 10⁻⁴ M and Na⁺ concentrations from 10⁻³ M to 0.15 M.
• A mathematical model was applied to deconvolute the fluorescence response into contributions from intercalative binding (high P/D) and electrostatic binding (low P/D).
• The method accounts for non-fluorescent dye associate formation on DNA during electrostatic binding, which quenches fluorescence.
• Binding site sizes were calculated by fitting the titration data to models assuming independent, non-cooperative binding with defined site lengths.
• The contribution of each binding mode to total complex formation was quantified as a function of P/D ratio using the fluorescence anisotropy and intensity data.

Experimental results

Research questions

• RQ1 What are the relative contributions of intercalative and electrostatic binding modes to quinacrine-DNA complex formation at different P/D molar ratios?
• RQ2 How does ionic strength influence the competition between intercalative and electrostatic binding modes?
• RQ3 What is the binding site size for each mode, and how does it affect the competitive dominance of electrostatic binding despite lower affinity?
• RQ4 How does the formation of non-fluorescent dye associates on DNA affect the observed fluorescence intensity during electrostatic binding?
• RQ5 Can polarized fluorescence data be used to quantitatively deconvolute the contributions of multiple binding modes to DNA-ligand complexation?

Key findings

• The minimum in the relative fluorescence intensity vs. P/D titration curve arises from competition between intercalative binding (dominant at high P/D) and electrostatic binding (dominant at low P/D).
• Electrostatic binding is accompanied by the formation of non-fluorescent dye associates on the DNA backbone, which quench fluorescence and contribute to the observed minimum in the titration curve.
• The binding site size for electrostatic binding is 8 times smaller than that for intercalative binding, explaining its competitive advantage at low P/D despite weaker affinity.
• The contribution of electrostatic binding to total complex formation exceeds that of intercalation at low P/D ratios, even though intercalation has a higher intrinsic binding affinity.
• The method presented allows for quantitative calculation of the fraction of each binding mode in the complex as a function of P/D, enabling detailed thermodynamic and kinetic analysis.
• The study demonstrates that fluorescence anisotropy and intensity data can be used to resolve multiple binding modes in DNA-ligand systems when combined with appropriate modeling.