Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

Synthesis, Characterization and DNA-Binding Affinity of a New Zinc(II) Bis(5-methoxy-indol-3-yl)propane-1,3-dione Complex.

The novel zinc(II) µ-oxo-bridged-dimeric complex [Zn2(µ-O)2(BMIP)2] (BMIP = 1,3-bis(5-methoxy-1-methyl-1H-indol-3-yl)propane-1,3-dione), 1, was synthetized and fully characterized. The spectral data indicate a zincoxane molecular structure, with the BMIP ligand coordinating in its neutral form via its oxygen atoms. Structural changes in 1 in dimethylsulfoxide (DMSO) were evidenced by means of spectroscopic techniques including infrared absorption and nuclear magnetic resonance, showing DMSO entrance in the coordination sphere of the metal ion. The resulting complex [Zn2(µ-O)2(BMIP)2(DMSO)], 2, readily reacts in the presence of N-methyl-imidazole (NMI), a liquid-phase nucleoside mimic, to form [Zn2(µ-O)2(BMIP)2(NMI)], 3, through DMSO displacement. The three complexes show high thermal stability, demonstrating that 1 has high affinity for hard nucleophiles. Finally, with the aim of probing the suitability of this system as model scaffold for new potential anticancer metallodrugs, the interactions of 1 with calf thymus DNA were investigated in vitro in pseudo-physiological environment through UV-Vis absorption and fluorescence emission spectroscopy, as well as time-resolved fluorescence studies. The latter analyses revealed that [Zn2(µ-O)2(BMIP)2(DMSO)] binds to DNA with high affinity upon DMSO displacement, opening new perspectives for the development of optimized drug substances.

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.