ABSTRACT
The formation of a stable G-quadruplex (GQ), a noncanonical DNA secondary structure, can inhibit the elevated telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most of the biochemical processes involving GQs might require local conformational changes at the guanine tetrad (G4) level. These local conformational changes of individual G4 layers during protein and drug interactions have not yet been explored in detail. This is because the spectroscopic signals of individual G4 layers are concealed in the total signal of GQ. Here we report a method to study the local conformations of individual G4 layers in GQs that uses 6-methylisoxanthopterine (6MI), a Circular Dichroism (CD)-active fluorescent base analogue of guanine. A synthetic, tetra molecular, parallel GQ with site-specifically positioned 6MI monomers or dimers was used as the substrate. Analytical ultracentrifugation studies and gel electrophoretic studies showed that properly positioned 6MI monomers and dimers could form stable GQs with CD-active fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then tracked by following the incorporated probes absorbance, fluorescence intensity, and circular dichroism changes. Further, thermal melting and acrylamide fluorescence quenching experiments were performed to confirm the stability and solvent accessibility of individual G4 layers. Overall, the study showed that site-specifically incorporated CD active fluorescent base analogues could be used as a probe to monitor the local conformational changes of individual G4 layers of a GQ structure. This method can be applied to explore the details of small molecule-GQ interaction at the G4 layer, which will be useful in designing drugs for GQ-related genetic disorders, cancer, and aging.
Competing Interest Statement
The authors have declared no competing interest.
