The energy landscapes of human telomeric G-quadruplexes are complex, and their folding pathways have remained largely unexplored. By using real-time NMR spectroscopy, we investigated the K(+)-induced folding of the human telomeric DNA sequence 5'-TTGGG(TTAGGG)3 A-3'. Three long-lived states were detected during folding: a major conformation (hybrid-1), a previously structurally uncharacterized minor conformation (hybrid-2), and a partially unfolded state. The minor hybrid-2 conformation is formed faster than the more stable hybrid-1 conformation. Equilibration of the two states is slow and proceeds via a partially unfolded intermediate state, which can be described as an ensemble of hairpin-like structures.

G-quadruplex structures can be formed at the single-stranded overhang of telomeric DNA, and ligands able to stabilize this structure have recently been identified as potential anticancer drugs. Among the potential G-quadruplex binders, we have studied the binding ability of berberine and sanguinarine, two members of the alkaloid family, an important class of natural products long known for medicinal purpose. Our spectroscopic (CD, NMR, and fluorescence) studies and molecular modeling approaches revealed binding modes at ligand-complex stoichiometries >1:1 and ligand self-association induced by DNA for the interactions of the natural alkaloids berberine and sanguinarine with the human telomeric G-quadruplex DNA.

The structural differences among different G-quadruplexes provide an opportunity for site-specific targeting of a particular G-quadruplex structure. However, majority of G-quadruplex ligands described thus far show little selectivity among different G-quadruplexes. In this work, we delineate the design and synthesis of a crescent-shaped thiazole peptide that preferentially stabilizes c-MYC quadruplex over other promoter G-quadruplexes and inhibits c-MYC oncogene expression. Biophysical analysis such as Förster resonance energy transfer (FRET) melting and fluorescence spectroscopy show that the thiazole peptide TH3 can selectively interact with the c-MYC G-quadruplex over other investigated G-quadruplexes and duplex DNA. NMR spectroscopy reveals that peptide TH3 binds to the terminal G-quartets and capping regions present in the 5′- and 3′-ends of c-MYC G-quadruplex with a 2:1 stoichiometry; whereas structurally related distamycin A is reported to interact with quadruplex structures via groove binding and end stacking modes with 4:1 stoichiometry. Importantly, qRT-PCR, western blot and dual luciferase reporter assay show that TH3 downregulates c-MYC expression by stabilizing the c-MYC G-quadruplex in cancer cells. Moreover, TH3 localizes within the nucleus of cancer cells and exhibits antiproliferative activities by inducing S phase cell cycle arrest and apoptosis.

A carbazole derivative (BTC) regulates the dynamics of unstructured human c-MYC and h-TELO sequences by folding them into compact quadruplex structures.