G-quadruplexes (G4s), which are structures formed in guanine-rich regions of DNA, are involved in a variety of significant biological functions, and therefore “sequence-dependent” selective G4-stabilizing agents are required as tools to investigate and modulate these functions. Here, we describe the synthesis of a new series of macrocyclic hexaoxazole-type G4 ligand (6OTD) bearing three side chains. One of these ligands, 5b, stabilizes telomeric G4 preferentially over the G4-forming DNA sequences of c-kit and K-ras, due to the interaction of its piperazinylalkyl side chain with the groove of telomeric G4.

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.

Oxazole-type fluorophores show an increase of fluorescence intensity upon interaction with nucleic acids, and therefore can be used as tools for nucleic acid-sensing and fluorescence imaging. Here, we developed a novel stilbene-type fluorophore, MO-VN (1), consisting of a mono oxazole bearing a vinyl naphthalene moiety. This compound (1) was embedded in a trioxazole 2 and a cyclic hexaoxazole 3a. The fluorescence properties of 1, 2, and 3a were evaluated in the presence of various nucleic acid sequences. Compound 3 showed significant fluorescent enhancement upon interacting with G-quadruplex (G4) structure, which plays critical roles in various biological phenomena. Further structural development focusing on the vinyl naphthalene moiety of 3a afforded a turn-on type G4 ligand 3e that shows G4-specific fluorescence. Measurement of the fluorescence of 3e during titration of a telomeric DNA, telo24, with its C-rich complementary sequence, which unwinds the G4 structure, allowed us to monitor the dynamics of G4.

We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC₃) on intermolecular G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures, has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.