DNA quadruplexes (G4s) are highly conserved secondary structures that form in guanine-rich sections of chromosomal DNA, particularly within telomeric and promoter regions. Due to their involvement in processes such as aging and cancer, they have emerged as suitable targets in various areas of research. A common strategy involves the selective stabilization of G4 structures to inhibit oncogene expression. However, G4s located within gene promoter regions often exhibit considerable inherent stability, approaching the limits of melting assays and single-molecule force spectroscopy. The oncogene promoter c-KIT1 forms an exceptionally stable G4 structure, which previously lead to challenges in optical tweezers experiments due to the ∼40–50 pN force limitations imposed by conventional biotin/streptavidin and digoxigenin/antidigoxigenin dumbbell assays. By expanding the classic set-up with orthogonal click-chemistry strategies, we increased the applicable force range to ∼60 pN. This enabled reproducible characterization of the c-KIT1 quadruplex, including its folding and unfolding kinetics. Overall, this enhanced coupling approach broadens the applicability of single-molecule force spectroscopy to previously inaccessible, mechanically stable biomolecules, also beyond the field of DNA quadruplexes.
Wrobel et al. (Sun,) studied this question.