Stabilization of G-Quadruplex-Duplex Hybrid Structures Induced by Minor Groove-Binding Drugs

Abstract

Once it had been realized that G-quadruplexes exist in the cell and are involved in regulation of genomic processes, the quest for ligands recognizing these noncanonical structures was underway. Many organic compounds that tightly associate with G-quadruplexes have been identified. However, the specificity of G-quadruplex-binding ligands towards individual structures remains problematic, as the common recognition element of these ligands is the G-tetrad. In this paper, we focus on G-quadruplex-duplex hybrids (QDH) containing a hairpin duplex incorporated as a stem-loop into the G-quadruplex core. The presence of a stem-loop renders QDH amenable to sequence-specific recognition by duplex-binding drugs. Should the thermodynamic crosstalk between the stem-loop and the tetraplex core be sufficiently strong, the drug binding to the loop would lead to the stabilization of the entire structure. We studied the stabilizing influence of the minor groove-binders netropsin and Hoechst 33258 on a family of QDH structures, as well as a G-quadruplex and a hairpin modeling the G-quadruplex core and the stem-loop of the QDH’s. We found that the binding of either drug results in an enhancement of the thermal stability of all DNA structures, as expressed by increases in the melting temperature, TM. Analysis of the hierarchical order of increases in TM revealed that the drug-induced stabilization arises from drug binding to the G-quadruplex domain of a QDH and the stem-loop, if the latter contains an all-AT binding site. This result attests to the thermodynamic crosstalk between the stem-loop and the tetraplex core of a QDH. Given the existing library of minor groove-binding drugs recognizing mixed A·T and G·C DNA sequences, our results point to an untapped avenue for sequence-specific recognition of QDH structures in vitro and, possibly, in vivo; thereby, opening the way for selective stabilization of four-stranded DNA structures at predetermined genomic loci, with implications for the control of genomic events.