Guanine-rich tracts are observed in critical segments of eukaryotic genomes including telomeric, intronic and oncogenic promotor DNA regions, as well as within 5'-untranslated regions (UTRs) of oncogenic RNA transcripts. Such putative G-quadruplex-forming sequences are prevalent in proto-oncogenes (which promote cell proliferation) and essentially lacking in tumor-suppressor genes (which maintain genomic stability). Cellular proteins exist that bind, cleave, resolve, promote and disrupt G-quadruplex formation, with recent research providing increasing support for G-quadruplex formation in vivo. Our laboratory has ongoing projects aimed at NMR and x-ray structural characterization of G-quadruplex topologies formed by guanine-rich tracts in c-myc, c-kit, VEGF and c-RET oncogenic DNA promoters (Aim 1), in human telomeric and intronic DNA (Aim 2), and in N-ras, and related oncogenic RNA 5'-UTR segments (Aim 3). These structural studies should define the folding propensity and diversity of G-quadruplex topologies, as well as the energetics of interconversion between conformational states. The research will be extended to structural characterization of G-quadruplex-duplex junctions and to simplified models of telomeric t-loops, where the telomeric 3'-ovehang is protected through invasion into an adjacent duplex segment. Ligand-induced stabilization of telomeric G-quadruplex scaffolds in humans, resulting in the inhibition of telomerase activity, constitutes a promising strategy for anti-cancer drug development. We propose to structurally characterize complexes of ligands exhibiting unique selectivity towards telomeric, intronic, oncogenic promotor and 5'-UTR G-quadruplexes identified in our laboratory (Aim 4), thereby providing structural insights into the action of potent inhibitors of telomerase and oncogene regulation at the level of transcription (promoters) and translation (5'- UTR). Our initial efforts are focused on oxazole-containing macrocycles, analogs of telomestatin, the most potent inhibitor of telomerase, but will be expanded to promising small-molecule shape-sensitive G-quadruplex-interacting ligands. There are no structures reported in the literature of G-quadruplexes bound to peptides and proteins. To address this issue (Aim 5), we are currently undertaking the NMR-based structural characterization of the complex between L-vasopressin and an in vitro selected mirror-image 38-mer L-RNA aptamer (spiegelmer) that folds into a G-quadruplex scaffold.
Four-stranded nucleic acid scaffolds, designated G-quadruplexes are formed at guanine-rich tracts of oncogenic promoters, telomeres and intronic sequences, as well as 5'-untranslated regions of RNAs. Small molecule ligands can target and discriminate amongst the diverse sequence-dependent topologies of G-quadruplexes, thereby stabilizing their conformations and impacting on gene regulation at the transcriptional and translational levels. This application applies primarily NMR methods to determine the structures of G-quadruplex folds and their ligand complexes to decipher rules governing higher-order nucleic acid architecture and the principles dictating molecular recognition, with promising impact on human health, as reflected for instance, in the potential of stabilized G-quadruplexes generated at telomeric ends in inhibiting the activity of telomerase, an enzyme upregulated in tumor cells.
|Lim, Kah Wai; Amrane, Samir; Bouaziz, Serge et al. (2009) Structure of the human telomere in K+ solution: a stable basket-type G-quadruplex with only two G-tetrad layers. J Am Chem Soc 131:4301-9|
|Patel, D J; Shapiro, L; Kozlowski, S A et al. (1986) Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex. Biochemistry 25:1036-42|
|Patel, D J; Shapiro, L; Kozlowski, S A et al. (1986) Structural studies of the O6meG.C interaction in the d(C-G-C-G-A-A-T-T-C-O6meG-C-G) duplex. Biochemistry 25:1027-36|
|Patel, D J; Shapiro, L (1986) Sequence-dependent recognition of DNA duplexes: netropsin complexation to the TATA site of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution. Biopolymers 25:707-27|
|Patel, D J; Shapiro, L; Hare, D (1986) Sequence-dependent conformations of DNA duplexes: the TATA segment of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution. Biopolymers 25:693-706|
|Patel, D J; Shapiro, L; Hare, D (1986) Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution. J Biol Chem 261:1223-9|
|Patel, D J; Shapiro, L (1986) Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution. J Biol Chem 261:1230-40|
|Patel, D J; Shapiro, L; Kozlowski, S A et al. (1986) Covalent carcinogenic O6-methylguanosine lesions in DNA. Structural studies of the O6 meG X A and O6meG X G interactions in dodecanucleotide duplexes. J Mol Biol 188:677-92|
|Patel, D J; Shapiro, L (1985) Molecular recognition in noncovalent antitumor agent-DNA complexes: NMR studies of the base and sequence dependent recognition of the DNA minor groove by netropsin. Biochimie 67:887-915|
|Patel, D J; Shapiro, L; Kozlowski, S A et al. (1985) Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes. Biochimie 67:861-86|