Kinetic resolution of bimolecular hybridization versus intramolecular folding in nucleic acids by surface plasmon resonance: application to G-quadruplex/duplex competition in human c-myc promoter

The human oncogene c-myc is regulated by G-quadruplex formation within the nuclease hypersensitive element (NHE III(I)) in the c-myc promoter, making the quadruplex a strong anti-cancer target. With respect to this, the competing equilibrium between intramolecular quadruplex folding and bimolecular duplex formation is poorly understood and very few techniques have addressed this problem. We present a method for simultaneously determining the kinetic constants for G-quadruplex folding/unfolding and hybridization in the presence of the complementary strand from a single reaction using an optical biosensor based on surface plasmon resonance (SPR). Using this technique, we demonstrate for the first time that quadruplex formation in the c-myc promoter is favored at low strand concentrations. Our results indicate favorable quadruplex folding (equilibrium folding constant K(F) of 2.09 calculated from the kinetic parameters: folding rate constant, k(f) = 1.65 x 10(-2) s(-1) and unfolding rate constant, k(u) = 7.90 x 10(-3) s(-1)) in 150 mM K+. The hybridization rate constants detected concurrently gave a bimolecular association constant, k(a) = 1.37 x 10(5) M(-1) s(-1) and dissociation constant, k(d) = 4.94 x 10(-5) s(-1). Interestingly, in the presence of Na+ we observed that G-quadruplex folding was unfavorable (K(F) = 0.54). Implication of our results on the c-myc transcription activation model is discussed in light of aberrant c-myc expression observed on destabilization of the G-quadruplex.