Quantitative Effects of the Loop Region on Topology, Thermodynamics, and Cation Binding of DNA G-quadruplexes.

ABSTRACT

The thermal stability of G-quadruplexes is important for their biological roles. G-quadruplexes are stable in the presence of cations such as K+ and Na+ because these cations coordinate in the G-quartet of four guanine bases. It is well known that the number of G-quartets and the configuration of the guanine bases affect the binding affinity of the cation. Recently, structures formed in the loop regions connecting the guanine stretches have attracted significant attention, because the loop region affects G-quadruplex properties, such as topology, thermal stability, and interactions with proteins and small molecules. Considering these effects, the loop region can also affect the binding affinity of the cations. Here, we designed a series of G-quadruplex-forming DNA sequences that contain a hairpin in a loop region and investigated the effects of the sequence and structure of the loop region on the cation binding affinity as well as the thermal stability of the G-quadruplex as a whole. First, structural analysis of the DNA sequences showed that the hairpin at the loop plays a key role in determining G4 topology (strand orientation). Second, in the case of the G-quadruplexes with the hairpin-forming loop region, it was found that a longer loop length led to a higher thermodynamic stability of the G-quadruplex as well as higher cation binding affinity. In contrast, an unstructured loop region did not lead to such effects. Interestingly, the cation binding affinity was correlated to the thermodynamic stability of the hairpin structure at the loop region. It was quantitatively demonstrated that the stable loop region stabilized the whole G-quadruplex structure, which induced higher cation binding affinity. These systematic and quantitative results showed that the loop region is one of the determinants of cation binding and expanded the possibilities of drug development targeting G4s by stabilizing the loop region.