Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation

Abstract

G-quadruplex (G4) selective stabilizing ligands can regulate c-MYC gene expression, but the kinetic basis remains unclear. Determining the effects of ligands on c-MYC promoter G4s' folding/unfolding kinetics is challenging due to the polymorphic nature of G4s and the high energy barrier to unfold c-MYC promoter G4s. Here, we used single-molecule magnetic tweezers to manipulate a duplex hairpin containing a c-MYC promoter sequence to mimic the transiently denatured duplex during transcription. We measured the effects of six commonly used G4s binding ligands on the competition between quadruplex and duplex structures, as well as the folding/unfolding kinetics of G4s. Our results revealed two distinct roles for G4s selective stabilization: CX-5461 is mainly acting as c-MYC G4s stabilizer, reducing the unfolding rate (k u ) of c-MYC G4s, whereas PDS and 360A also act as G4s chaperone, accelerating the folding rates (k f ) of c-MYC G4s. qRT-PCR results obtained from CA46 and Raji cell lines demonstrated that G4s stabilizing ligands can downregulate c-MYC expression, while G4s stabilizer CX-5461 exhibited the strongest c-MYC gene suppression. These results shed light on the potential of manipulating G4s' folding/unfolding kinetics by ligands for precise regulation of promoter G4-associated biological activities.

Unfolding rates of 1:1 and 2:1 complex of CX-5461 and c-<i>MYC</i> promoter G-quadruplexes revealed by single-molecule force spectroscopy

“…The single-stranded DNA (ssDNA) c- MYC G4s forming sequence was labeled with a carboxyfluorescein (FAM) fluorophore at either the 5'-end or 3'-end and titrated with different concentrations of CX-5461. CX-5461 does not directly quench the fluorescein dye at a concentration of 1 μmol/L (Zhang et al 2024 ), but upon binds to c- MYC G4s, CX-5461 induced quenching of FAM fluorescence in both 5' and 3' FAM-labeled c- MYC G4s, indicating its ability to bind at both ends. Thus, the dissociation constant K d of CX-5461 to the binding site on each end of c- MYC G4s can be determined by analyzing the changes in fluorescence intensity.…”

“…The impacts of CX-5461 on the unfolding forces of c- MYC G4s were characterized using single-molecule magnetic tweezers to unfold a short dsDNA hairpin that contains a c- MYC promoter G4 forming sequences and complementary strand ( Fig. 2A ) (Zhang et al 2024 ). The Myc-hairpin DNA was ligated with two double-stranded DNA handles (489 bp and 609 bp) and was tethered between a streptavidin-coated paramagnetic bead and a coverslip.…”

“…Single-molecule force spectroscopy (SMFS) is a powerful tool for investigating the folding and unfolding kinetics of c- MYC promoter G4s under physiologically relevant conditions (Cheng et al 2020 ; You et al 2015 ). This technique allows us to quantify how proteins (Cheng et al 2021 ; You et al 2017 ) and small ligands (Zhang et al 2024 ) influence the folding/unfolding rates of c- MYC G4s. We recently employed SMFS with magnetic tweezers and demonstrated that CX-5461 is a strong G4s stabilizer, which significantly increases the average unfolding forces of c-MYC G4s at physiologically relevant potassium concentrations (Zhang et al 2024 ).…”

“…This technique allows us to quantify how proteins (Cheng et al 2021 ; You et al 2017 ) and small ligands (Zhang et al 2024 ) influence the folding/unfolding rates of c- MYC G4s. We recently employed SMFS with magnetic tweezers and demonstrated that CX-5461 is a strong G4s stabilizer, which significantly increases the average unfolding forces of c-MYC G4s at physiologically relevant potassium concentrations (Zhang et al 2024 ). While average unfolding forces provide valuable information about the G4s stabilization, they don’t quantify the unfolding rates of CX-5461-G4 complexes.…”

“…Here, using our recently developed single-molecule manipulation methods (Zhang et al 2024 ), we conducted further measurements of the unfolding-force distributions of c- MYC G4s in the presence of different concentrations of CX-5461 ( Fig. 1A ).…”