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The G-quadruplex (G4) is a four-stranded DNA structure identified in vivo in guanine-rich regions located in the promoter of a number of genes. Intriguing evidence suggested that small molecules acting as G4-targeting ligands could potentially regulate multiple cellular processes via either stabilizing or disruptive effects on G4 motifs. Research in this field aims to prove the direct role of G4 ligands and/or structures on a specific biological process in a complex living organism. In this study, we evaluate in vivo the effects of a nickel(II)-salnaphen-like complex, named Nisaln, a potent G4 binder and stabilizer, during embryogenesis of the sea urchin embryo. We describe developmental defects inflicted by Nisaln and correlate them with variation in the expression of several regulatory genes. It is worth mentioning that we show that Nisaln binds a G4 structure in the promoter of hbox12-a, a gene lying at the top of the developmental regulatory hierarchy, inducing overexpression of this gene.

The interaction of doxorubicin (DX) with model polynucleotides poly(dG-dC)·poly(dG-dC) (polyGC), poly(dA-dT)·poly(dA-dT) (polyAT), and calf thymus DNA has been studied by several spectroscopic techniques in phosphate buffer aqueous solutions. UV-vis, circular dichroism, and fluorescence spectroscopic data confirm that intercalation is the prevailing mode of interaction, and also reveal that the interaction with AT-rich regions leads to the transfer of excitation energy to DX not previously documented in the literature. Moreover, the DX affinity for AT sites has been found to be on the same order of magnitude as that reported for GC sites.

Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL-1. Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1. Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.

A proline, a leucine, and two cysteine residues, introduced at positions 19, 28, 31, and 32 of bovine pancreatic RNase A, i.e. the positions occupied by these residues in the subunit of bovine seminal RNase, the only dimeric RNase of the pancreatic-type superfamily, transform monomeric RNase A into a dimeric RNase, endowed with the same ability of BS-RNase of swapping its N-terminal segments. The thermodynamic consequences of the progressive introduction of these four residues into RNase A polypeptide chain have been studied by comparing the temperature- and urea-induced denaturation of three mutants of RNase A with that of a stable monomeric derivative of BS-RNase. The denaturation processes proved reversible for all proteins, and well represented by the two-state N--D transition model. The progressive introduction of the four residues into RNase A led to a gradual shift of the protein stability toward that characteristic of monomeric BS-RNase, which, in turn, is markedly less stable than RNase A with respect to both temperature- and urea-induced denaturation. On the other hand, the thermal stability of a dimeric active mutant of RNase A is found to approach that of wild-type seminal RNase.