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

Area of Science:

• Molecular Biology
• Biophysics

Background:

• G-quadruplex (G4) selective stabilizing ligands influence c-MYC gene expression.
• The kinetic mechanisms underlying this regulation are not fully understood.
• Investigating ligand effects on c-MYC promoter G4s' folding/unfolding kinetics is complex due to G4 polymorphism and high unfolding energy barriers.

Purpose of the Study:

• To elucidate the kinetic basis of how G4 selective stabilizing ligands regulate c-MYC gene expression.
• To measure the impact of commonly used G4 binding ligands on c-MYC promoter G4s' folding and unfolding kinetics.
• To understand the competition between quadruplex and duplex structures in the presence of these ligands.

Main Methods:

• Utilized single-molecule magnetic tweezers to manipulate a duplex hairpin containing a c-MYC promoter sequence.
• Mimicked transiently denatured duplexes during transcription to study G4 dynamics.
• Measured folding/unfolding kinetics and ligand-induced structural competition for six G4 binding ligands.

Main Results:

• Identified two distinct roles for G4 selective stabilization: CX-5461 acts as a stabilizer, reducing G4 unfolding rates (k_u).
• PDS and 360A function as chaperones, accelerating G4 folding rates (k_f).
• In cell lines (CA46, Raji), G4 ligands downregulated c-MYC expression, with CX-5461 showing the strongest suppression.

Conclusions:

• Ligand-mediated manipulation of G-quadruplex folding/unfolding kinetics offers a strategy for precise regulation of promoter G4-associated biological activities.
• CX-5461's primary role as a stabilizer and PDS/360A's chaperone activity provide mechanistic insights into c-MYC regulation.
• This study highlights the potential therapeutic applications of G4 ligands in controlling gene expression.