G-Quadruplex Unwinding Molecular Mechanisms by Helicases and Their Applications

G-quadruplexes (G4s) are specialized nucleic acid structures extensively formed throughout the genome, with particular enrichment in regulatory regions such as telomeres, promoters, and transcriptional enhancers. These four-stranded assemblies are involved in multiple chromosomal processes, including DNA replication, transcription, maintenance of genomic stability, and epigenetic regulation, and are closely associated with cancer biology. Due to their unusual thermodynamic stability, G4s serve as physical barriers to DNA/RNA unwinding, thereby impeding replication, transcription, and translation and compromising genome integrity. To mitigate this threat, cells have evolved dedicated helicases that can actively resolve G4 structures. In this review, we summarize recent structural advances-primarily derived from protein crystallography-regarding the mechanisms by which helicases unwind G4 quadruplexes. The insights presented herein establish a framework for elucidating the molecular basis of G4 unfolding and for the rational design of small-molecule G4 ligands and therapeutic agents. Additionally, we explore the applications of G4 helicases in nanopore sequencing, which aim to enhance sequencing accuracy, throughput, and continuity.