Mechanistic insights into the G-quadruplex-duplex equilibrium in the c-kit promoter: Role of ions and flanking sequences

Abstract

Nucleic acid-ion interactions constitute an intrinsic cornerstone of the physicochemical, energetic, and structural attributes of DNA and RNA. These interactions determine the folding and stabilization of intricate secondary and tertiary architectures, such as G-quadruplexes (GQs), which have pivotal functional significance in telomeric regions, origins of replication, and various gene promoter sequences. Earlier studies have elucidated that the 22-mer c-kit promoter sequence adopts a parallel-stranded G-quadruplex conformation, exhibiting a high degree of structural conservation within its duplex and quadruplex form. However, detailed mechanistic insight into this interconversion of these two conformations at single-molecule resolution has not been explored explicitly. In this work we have employed single-molecule FRET experiments (smFRET) to investigate the dynamic interconversion between the usual Watson-Crick duplex structure and the G-quadruplex structure. Herein, we assess the sensitivity of the G quadruplex-duplex equilibrium by introducing a short complementary strand as a pharmacological agent in the presence of Na⁺/K⁺ ions. This subtle mechanistic detail of the opening of the quadruplex, because of the hybridization with the complementary strand, reveals that the opening of the K⁺-stabilised G-quadruplex is slower than the opening of the Na⁺-stabilised form. smFRET along with CD and ensemble fluorescence measurements demonstrated the ion-mediated effect on the stability and equilibrium of the G-quadruplex and duplex structure. Furthermore, we explored the influence of flanking bases on quadruplex stability and duplex formation in the c-kit promoter region, providing a detailed analysis of how these factors modulate structural transitions.