Roles of Loop Region in Folding Kinetics and Transcription Inhibition of DNA G-Quadruplexes

Targeting G-quadruplexes, which have distinctive structures, to regulate biological reactions in cells has attracted interest due to the many disease-related genes that possess G-quadruplex-forming sequences. To achieve regulation of gene expression using G-quadruplexes, their folding kinetics and time scales should be well understood. However, the G-quadruplex folding kinetics is highly dependent on its nucleotide sequence as well as its surrounding environment, and thus a general folding mechanism is difficult to propose. Moreover, the effects of G-quadruplex folding kinetics on biological functions such as transcription inhibition are not represented yet. Here, we investigated the folding kinetics and mechanism of G-quadruplexes by focusing on the loop region. Kinetic analyses showed that the hairpin structure in the second loop region significantly accelerated G4 folding, suggesting that it served as a nucleation site for the subsequent folding process. The hairpin in the second loop adopted an intermediate state, an antiparallel G4 structure, in the folding process. Moreover, T7 polymerase assay demonstrated that faster G4 folding resulted in more efficient transcription inhibition. These findings demonstrate the importance of hairpin in the G4 folding kinetics and mechanism and a new strategy for developing G4-targeting small molecules.