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

Nucleic Acids Res. 2005 Aug 5;33(14):4466-74. doi: 10.1093/nar/gki750. Print 2005.

Abstract

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / chemistry*
  • G-Quadruplexes
  • Genes, myc*
  • Guanine / chemistry
  • Humans
  • Kinetics
  • Nucleic Acid Conformation
  • Nucleic Acid Hybridization / methods*
  • Promoter Regions, Genetic*
  • Purines / chemistry
  • Surface Plasmon Resonance / methods*
  • Transcriptional Activation

Substances

  • Purines
  • Guanine
  • DNA