The nuclease hypersensitivity element III1 (NHE III1) upstream of the P1 and P2 promoters of c-MYC controls 80-90% of the transcriptional activity of this gene. The purine-rich strand in this region can form a G-quadruplex structure that is a critical part of the silencer element for this promoter. We have demonstrated that this G-quadruplex structure can form a mixture of four biologically relevant parallel-loop isomers, which upon interaction with the cationic porphyrin TMPyP4 are converted to mixed parallel/antiparallel G-quadruplex structures.

The human telomeric sequence d[T(2)AG(3)](4) has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na(+)), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K(+)). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K(+) or Na(+). In contrast, in the presence of K(+), Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures.

The proximal 5′-flanking region of the human platelet-derived growth factor A (PDGF-A) promoter contains one nuclease hypersensitive element (NHE) that is critical for PDGF-A gene transcription. On the basis of circular dichroism (CD) and electrophoretic mobility shift assay (EMSA), we have shown that the guanine-rich (G-rich) strand of the DNA in this region can form stable intramolecular parallel G-quadruplexes under physiological conditions. A Taq polymerase stop assay has shown that the G-rich strand of the NHE can form two major G-quadruplex structures, which are in dynamic equilibrium and differentially stabilized by three G-quadruplex-interactive drugs. One major parallel G-quadruplex structure of the G-rich strand DNA of NHE was identified by CD and dimethyl sulfate (DMS) footprinting. Surprisingly, CD spectroscopy shows a stable parallel G-quadruplex structure formed within the duplex DNA of the NHE at temperatures up to 100°C. This structure has been characterized by DMS footprinting in the double-stranded DNA of the NHE. In transfection experiments, 10 μM TMPyP4 reduced the activity of the basal promoter of PDGF-A ∼40%, relative to the control. On the basis of these results, we have established that ligand-mediated stabilization of G-quadruplex structures within the PDGF-A NHE can silence PDGF-A expression.

The components and cofactors of the holoenzyme telomerase and its substrate telomeric DNA are attractive targets for anticancer agents that act by inhibiting the activity of telomerase. This review outlines recent advances in telomerase inhibition that have been achieved using antisense oligonucleotides and ribozymes that target the telomerase mRNA or its hTR RNA template. Although these are potent catalytic inhibitors of telomerase, they are challenging to implement in the clinic due to their delayed effectiveness. Drugs that directly bind to the telomeres, the complex structures that are associated at the telomeric ends, and stabilize secondary DNA structures such as G-quadruplexes are also potent inhibitors of telomerase. Special focus is given here to the telomeres, the biological machinery that works in tandem with telomerase to elongate telomeres, the causes of telomere disruption or dysfunction, and the consequences of disruption/dysfunction on the activity and design of anticancer agents.