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

摘要

The human oncogene c-myc is regulated by G-quadruplex formation within the nuclease hypersensitive element (NHE IIII) 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 KF of 2.09 calculated from the kinetic parameters: folding rate constant, kf = 1.65 × 10⁻² s⁻¹ and unfolding rate constant, ku = 7.90 × 10⁻³ s⁻¹) in 150 mM K⁺. The hybridization rate constants detected concurrently gave a bimolecular association constant, ka = 1.37 × 10⁵ M⁻¹ s⁻¹ and dissociation constant, kd = 4.94 × 10⁻⁵ s⁻¹. Interestingly, in the presence of Na⁺ we observed that G-quadruplex folding was unfavorable (KF = 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.