The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

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A major control element of the human c-myc oncogene is the nuclease-hypersensitive purine/pyrimidine-rich sequence. This double-stranded DNA fragment, corresponding to the 27-base pair segment in the nucleasehypersensitive element of the c-myc promoter region, forms a stable Watson-Crick double helix under physiological conditions. However, this duplex DNA can be effectively converted to G-quadruplex DNA by a small molecular weight ligand. Both intermolecular and intramolecular G-quadruplex forms can be induced by this ligand. Similar transitional changes are also observed with the duplex telomeric sequence from the Oxytricha species. These results provide additional support to the idea that G-quadruplex structures may play structural roles in vivo and also provide insight into novel methodologies for rational drug design. These structurally altered DNA elements might serve as regulatory signals in gene expression or in telomere dynamics and hence are promising targets for drug action.The protein product of the c-myc protooncogene plays a vital role in the process of cellular growth and differentiation (1). Deregulation of c-myc expression has been detected in many cancers and is believed to be an important step in tumorigenesis (2). The control of c-myc gene expression is a complex process and occurs at various steps of transcription, such as initiation, elongation, and attenuation, as well as during the post-transcriptional stages. Although the mechanisms involved in this regulation are not yet completely understood, a major control element of the human c-myc oncogene has been localized. This is a purine/pyrimidine-rich region located 115 bases upstream from the P1 promoter, which controls up to 95% of the total c-myc transcription (3, 4). This DNA segment is highly sensitive to DNase I and S1 nuclease (5, 6) and is termed the nuclease-hypersensitive element (NHE).1 The appearance of this hypersensitive site is coupled with transcription activation of the c-myc oncogene. Structural variations in the NHE can influence the binding of transcription factors. Transcription factors such as heterogenous nuclear ribonucleoprotein K and nucleoside diphosphate kinase B (7) bind sequence specifically to the pyrimidine-rich strand of the NHE and activate c-myc transcription (8). The transacting factors heterogenous nuclear ribonucleoprotein A/B (9) and cellular nucleic acid-binding protein (10) bind to the NHE and are shown to augment c-myc expression in vitro. Apart from protein factors, antisense oligonucleotides bind to the NHE and repress c-myc transcription in vitro (11,12). Formation of a colinear triplex between the synthetic oligonucleotide and the NHE was proposed to cause this observed repression in transcription.The NHE has a high potential to form atypical DNA structures under superhelical stress. It was proposed to be in a slow equilibrium between a Watson-Crick base-paired double helix and an atypical DNA structure (6). Many models have been suggested to explain the conformational changes observed in the NHE...

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