Hoechst 33258 binds to G-quadruplex in the promoter region of human c-myc

Abstract

In vitro binding of Hoechst 33258 to the promoter region of human c-myc, d(GG GGAGGG TGG GGA GGG TGG GGA AGG TGG GG) which forms G-quadruplex, both in vitro and in vivo in the presence of metal ions was investigated by equilibrium absorption, fluorescence and kinetic surface plasmon resonance methods. Hypochromic effect in UV absorption spectra and blue shift in fluorescence emission maxima of Hoechst in the presence of quadruplex revealed that Hoechst binds to the quadruplex. Analysis of UV and fluorescence titration data revealed that Hoechst binds to quadruplex with binding affinity of the order of 10⁶. Anisotropy measurements and higher lifetime obtained from time-resolved decay experiments revealed that quadruplex-bound Hoechst is rotationally restricted in a less polar environment than the bulk buffer medium. From surface plasmon resonance studies, we obtained kinetic association (k_a) and dissociation (k_d) of 1.23 ± 0.04 × 10⁵ M⁻¹ s⁻¹ and 0.686 ± 0.009 s⁻¹, respectively. As Hoechst is known to bind A–T-rich region of duplex DNA, here we propose the likelihood of Hoechst interacting with the AAGGT loop of the quadruplex.