Osmolyte Changes the Binding Affinity and Mode of Interaction of Minor Groove Binder Hoechst 33258 with Calf Thymus DNA

Abstract

The bis-benzimidazole molecule Hoechst 33258 ( Fig. 1) is an important DNA minor groove binding ligand.1) Interaction of Hoechst 33258 has been extensively studied using oligonucleotides.2-5) Different aspects of this interaction viz., physicochemical, thermodynamics, NMR, X-ray crystallography, in silico simulation are now well established. 3,[6][7][8][9][10][11] From the classical observation on the ability of Hoechst 33258 binding in the AT rich region of minor groove of DNA. Synthetic chemists have designed various DNA minor groove binders for possible applications such as anticancer, antiviral, and other diseases. 12,13) Studies at cellular level have demonstrated the desired biological effects.14-16) New applications are emerging from studies related to radiosensitization and radioprotection in relation to modification of radiation response by Hoechst derivatives. [17][18][19][20][21] Attempts have also been made to correlate these observations with different Hoechst analogues.Physicochemical and thermodynamic studies have revealed important details on the mechanism of Hoechst 33258-DNA interaction. 12,[22][23][24] Role of water surrounding DNA has emerged as an important entity in the interaction processes. 12,22,25) The network of newly formed hydrogen bonds between the ligand and DNA replaces previously existing direct solute-solvent (water) hydrogen bonds in the minor groove. Hydration changes strongly influences the thermodynamics of ligand-DNA interaction. The change in hydration after the complex formation appeared to be governed by the structure of the ligand. 3,[26][27][28][29] Most of the studies and hence our understandings are still based on oligonucleotides. 5,27,30) Water molecules located close to the binding surfaces are more structured than those of bulk solvent. These locally structured water molecules are retained or released during a binding process with different thermodynamic consequences. Various techniques are available to study hydration changes accompanying ligand-DNA interaction 31,32) and osmotic stress approach has gained importance due to its ability to specifically alter water activity of the surrounding solution; a fact that has been harnessed to measure forces between molecules. 31,32) It is relatively a simple technique to study the hydration changes upon ligand-DNA interaction and gives stoichiometric amount of water release or uptake upon biomolecular complexation.27) The principle of osmotic stress method is based on Gibbs-Duhem equation. 34,35) The neutral solutes are added directly to the solution containing the ligand and macromolecule causes changes in the water activity of the solution. Osmotic stress method has been employed for studying the hydration changes for minor groove binder as well as for intercalators. 5,26,27,[36][37][38] In living system, small molecules interact with DNA in a very crowded environment. The presence of co-solutes in the in-vivo conditions affects the interaction process therefore it is important to study the interaction of Hoechst 33258...

In Vitro Assays Predictive of Telomerase Inhibitory Effect of G-Quadruplex Ligands in Cell Nuclei

“…In contrast, high selectivity of anionic ligands to the G-quadruplex over dsDNA has been reported, although the studies on anionic ligands have been only a few due to their lower affinity for G-quadruplexes than cationic ones. ,− Because of this high selectivity, anionic G-quadruplex ligands inhibit telomerase very efficiently, even in the presence of excess dsDNA. ,, Moreover, molecular crowding (MC), which mimics the high concentration of biomolecules in cells, , critically affects properties and reactions of biomolecules, including dsDNA , and G-quadruplex DNA, − by altering properties of the solution, including a decrease in the dielectric constant and water activity, ,, and an increase in viscosity and excluded volume . Systematic studies demonstrated that MC reduces binding between dsDNA and a variety of dsDNA ligands including intercalators and groove binders . Furthermore, Tan’s group recently reported that MC also reduces binding between the telomeric G-quadruplex and cationic G-quadruplex ligands, including TMPyP4, and their inhibition of telomerase .…”

Intrinsically Disordered Ku Protein-Derived Cell-Penetrating Peptides

“…Double-stranded ctDNA exhibits a negative Cotton effect at 245 nm corresponding to backbone helicity and a positive Cotton effect at 275 nm arising from nucleobase π–π stacking in B-conformation. As the peptide concentration increased, the ellipticity at 275 nm gradually decreased along with a concomitant redshift of the signal due to the interaction of peptides with ctDNA (Figure A, B). , The negative Cotton effect at 245 nm also moderately decreased with an increasing peptide concentration, indicating helicity distortion upon peptide binding. These results correlated with displacement assay data obtained by using the minor groove binding Hoechst dye.…”

Threading Intercalator-Induced Nanocondensates and Role of Endogenous Metal Ions in Decondensation for DNA Delivery

“…[C 50 of ligand] is the concentration of ligand at 50% reduction of Hoechst fluorescence. The calculated value of K Hoechst = 4.75 × 10 7 …”