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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...
Autores principales: | , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2005
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1183106/ https://www.ncbi.nlm.nih.gov/pubmed/16085756 http://dx.doi.org/10.1093/nar/gki750 |
Sumario: | 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 × 10(−2) s(−1) and unfolding rate constant, k(u) = 7.90 × 10(−3) s(−1)) in 150 mM K(+). The hybridization rate constants detected concurrently gave a bimolecular association constant, k(a) = 1.37 × 10(5) M(−1) s(−1) and dissociation constant, k(d) = 4.94 × 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. |
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