Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K+ solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K+ solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3′-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres.

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). Compounds that can stabilize the intramolecular DNA G-quadruplexes formed in the human telomeric sequence have been shown to inhibit the activity of telomerase and telomere maintenance, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. Knowledge of intramolecular human telomeric G-quadruplex structure(s) formed under physiological conditions is important for structure-based rational drug design and thus has been the subject of intense investigation. This review will give an overview of recent progress on the intramolecular human telomeric G-quadruplex structures formed in K + solution. It will also give insight into the structure polymorphism of human telomeric sequences and its implications for drug targeting.

We report the first G-quadruplex structure formed in the promoter region of the human bcl-2. Bcl-2 is a potent oncoprotein that functions as an inhibitor of cell apoptosis and has been found to be aberrantly overexpressed in a wide range of human tumors. A highly GC-rich region upstream of the P1 promoter plays an important role in the regulation of the transcriptional activity of the bcl-2 oncogene. The purine-rich strand of this region contains multiple runs of guanines and can form three distinct intramolecular G-quadruplexes in K + -containing solution. Of these, the G-quadruplex formed within the middle four consecutive guanine runs has been shown to be the most stable Gquadruplex structure, while it is also a mixture of loop isomers. This predominate G-quadruplex structure formed in this region was studied by NMR. Our results demonstrate a novel folding of a Email: yangd@pharmacy.arizona.edu. unique intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands. This Gquadruplex structure contains three G-tetrads connected with a single-nucleotide double-chainreversal side loop and two lateral loops. The three-nucleotide CGC loop in the bcl-2 promoter sequence forms a lateral loop, as opposed to a double-chain-reversal side loop observed in a similar sequence in the c-MYC promoter, which appears to largely determine the overall folding of the bcl-2 G-quadruplex. Furthermore, both the bcl-2 and c-MYC promoter sequences contain the G 3 NG 3 sequence motif, which forms a stable double-chain-reversal, parallel-stranded structural motif. This predominant bcl-2 G-quadruplex represents an attractive novel target for the design of new anticancer drugs that specifically modulate bcl-2 gene expression. NIH Public AccessBcl-2 (B-cell CLL/lymphoma 2) is a potent oncoprotein that plays an essential role in cell survival and functions as an inhibitor of cell apoptosis. 1 The bcl-2 proto-oncogene was first discovered in human follicular lymphoma and has been mapped to chromosome 18q21 based on a t(14;18) translocation to the immunoglobulin heavy chain (IgH) locus at 14q32. 2 Bcl-2 has been found to be aberrantly overexpressed in a wide range of human tumors, including Bcell and T-cell lymphomas, breast, prostate, cervical, colorectal, sand non-small cell lung carcinomas. 3 Elevation of bcl-2 level has also been associated with poor prognosis. 3 Thus the bcl-2 transcriptional control has emerged as an attractive target for anticancer therapeutics.The P1 promoter located 1386-1423 base pairs upstream of the translation start site is the major transcriptional promoter for bcl-2. 4 This is a TATA-less, GC-rich promoter that contains multiple transcriptional start sites. The 5′-end of the P1 promoter, including a highly GC-rich region, has been implicated in playing a major role in the regulation of bcl-2 transcription. 4 This GC-rich element is a 39-base-pair sequence that is located 58 to 19 base pairs upstream of the P1 promoter. Deletion or mutation of this element has been shown to increase prom...

Unimolecular parallel-stranded G-quadruplex structures are found to be prevalent in gene promoters. The nuclease hypersensitivity element III1 (NHE III1) of the c-MYC promoter can form transcriptionally active and silenced forms and the formation of DNA G-quadruplex structures has been shown to be critical for c-MYC transcriptional silencing. The solution structure of a 2:1 quindoline–G-quadruplex complex has been solved and shows unexpected features, including the drug-induced reorientation of the flanking sequences to form a new binding pocket. While both 3’ and 5’ complexes show overall similar features, there are identifiable differences which emphasize the importance of both stacking and electronic interactions. For the first time we describe the importance of the shape of the ligand as well as the two flanking bases in determining drug binding specificity. These structures provide important insights for the structure-based rational design of drugs that bind to unimolecular parallel G-quadruplexes commonly found in promoter elements.

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K+ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K+ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins.LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.