Influence of a Hairpin Loop on the Thermodynamic Stability of a DNA Oligomer

DSC was used to evaluate the mechanism of the thermally induced unfolding of the single-stranded hairpin HP = 5′-CGGAATTCCGTCTCCGGAATTCCG-3′ and its core duplex D (5′-CGGAATTCCG-3′)2. The DSC melting experiments performed at several salt concentrations were successfully described for HP and D in terms of a three-state transition model HP↔I (intermediate state) ↔ S (unfolded single-stranded state) and two state transition model D↔2S, respectively. Comparison of the model-based thermodynamic parameters obtained for each HP and D transition shows that in unfolding of HP only the HP↔I transition is affected by the TCTC loop. This observation suggests that in the intermediate state its TCTC loop part exhibits significantly more flexible structure than in the folded state while its duplex part remains pretty much unchanged

Modeling of Some Calorimetric and Spectropolarimetric Titration Data

Microcalorimetric and spectropolarimetric titrations were used to investigate micellization of cationic surfactants and binding of netropsin to dodecameric DNA duplex. For description of both processes, model functions containing linear and non-linear parameters were derived. Model analysis was based on a weighted (multi)linear regression and a standard »Simplex« procedure. Close investigation of the interplay of adjustable parameters has shown that a proper choice of the model function can significantly reduce the correlations between parameters. Values of physical properties (enthalpy changes, apparent equilibrium constants...) obtained from such curve modeling are in very good agreement with the corresponding values obtained by other methods. Equations and the calculation procedure reported here could be easily generalized and used for the description of some other concentration dependent properties in similar systems

Fluorescence studies of the effect of pH, guanidine hydrochloride and urea on equinatoxin II conformation

AbstractThe solvent denaturation of equinatoxin II (EgTxII) in aqueous solutions of urea, guanidine hydrochloride (Gu-HCI) and at various pH values was examined by monitoring changes in the protein intrinsic emission fluorescence spectra and in the fluorescence spectra of the added external probe ANS. It has been observed that EgTxII denaturation is reflected in a strong red shift of intrinsic fluorescence emission maxima accompanied by a simultaneous decrease in fluorescence intensity and that guanidine hydrochloride is significantly more powerful denaturant than urea or changing of pH. Comparison of intrinsic fluorescence spectra of EgTxII denatured by one of the three denaturing agents has shown that the fully denatured states of the protein in Gu-HCI and urea are similar and substantially different from those induced by changing of pH. Furthermore, according to the measurements of the ANS-fluorescence in EgTxII solutions as a function of pH the protein exists at pH values below 2.0 in an acid-denatured compact state

Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes

Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)(3) using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; K(Tel22) ≈ <K(dsDNA)) and for highly selective quadruplex-specific ligands (Phen-DC3, 360A-Br; K(Tel22) > K(dsDNA)). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation

What drives the binding of minor groove-directed ligands to DNA hairpins?

Understanding the molecular basis of ligand–DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule