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.
Halder
Kangkan
K
Institute of Genomics and Integrative Biology, CSIR Mall Road, Delhi 110007, India.
Chowdhury
Shantanu
S
D004247:DNA / Q000737:chemistry*; D054856:G-Quadruplexes; D016259:Genes, myc; D006147:Guanine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011401:Promoter Regions, Genetic; D011687:Purines / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D015533:Transcriptional Activation
D011687:Purines; D006147:Guanine; D004247:DNA