Guanine plus adenine-rich sequences can align through mismatch, triple, triad, tetrad, and hexad alignments, resulting in unique DNA architectures that differ in the number and orientation of strands and pairing alignments. Our research program applies NMR techniques to biologically relevant site-specific and uniformly 15N,13C-labeled DNA oligomer sequences to define the multistranded DNA architectures and their cation-dependent structural transitions, and to identify unique topologies associated with discriminatory recognition. We are also interested in defining the principles associated with ligand-G-quadruplex recognition, achieved either through stacking of planar ligands on terminal G-tetrads or through non-planar ligand recognition of base edges or groove dimensions. Three of our recent structural studies involving G-quadruplex scaffolds are outlined below and provide a flavor of ongoing higher order DNA architecture research in our laboratory.
Our laboratory has published the following reviews on G-quadruplexes:
Phan, A. T., Kuryavyi, V. & Patel, D. J. (2006). DNA architecture: from G to Z. Curr. Opin. Struct. Biol. 16, 288-298. [PubMed Abstract]
Phan, A. T., Kuryavyi, V., Luu, K. N. & Patel, D. J. (2006). “Structures of complex G-quadruplexes” in Quadruplex Nucleic Acids, (Neicdle, S. and Balasubramanian, S. Editors). RSC Biomolecular Sciences, Wiley, London, pp 81-99.
Patel, D. J., Phan, A. T. & Kuryavyi, V. (2007). Human telomere, oncogenic promotor and 5'-UTR G-quadruplexes: Diverse higher-order DNA and RNA targets for cancer therapeutics. Nucleic Acids Research 22, 7249-7255. [PubMed Abstract]
G-Quadruplexes formed by Telomeric Repeats
The terminal regions of eukaryotic chromosomes called telomeres are essential for stable chromosome maintenance. Human telomeric DNA is composed of (GGGTTA)n repeats, which forms G-quadruplex structures involving planar stacked G.G.G.G tetrads in Na or K cation-containing solution. Specifically, oligonucleotides containing one, two or four G-tracts have been shown to form tetrameric, dimeric or monomeric G-quadruplexes. These structures have recently become an attractive anticancer target impacting on telomerase function.
Four-Repeat Human Telomere forms (3+1) G-Quadruplex in K+ solution
Intermolecular G-quadruplexes formed by human telomere sequences are attractive anticancer targets. Recently, four-repeat human telomere sequences have been shown to form two different intermolecular (3+1) G-quadruplexes in K+ solution (Form 1 and form 2). We have solved the NMR-based solution structures of both Form 1 and Form 2 G-quadruplexes adopted by natural human telomere sequences. Both structures contain the (3+1) G-tetrad core with one double-chain-reversal loop and two edgewise loops, but differ in the successive order of loop arrangements within the G-quadruplex scaffold. Our structures provide the structural details at the two ends of the G-tetrad core in the context of natural sequences and information on different loop conformations.
Luu, K. N., Phan, A. T., Kuryavyi, V., Lacroix, L. & Patel, D. J. (2006). Structure of the human telomere in K+ solution: An intramolcular (3+1) G-quadruplex scaffold. J. Am. Chem. Soc. 128, 9963-9970. [PubMed Abstract]
Phan, A. T., Kuryavyi, V., Luu. K. N. & Patel, D. J. (2007). Structure of two intramolecular G-quadruplexes formed by natural human telomere sequences in K+ solution. Nucleic Acids Research 35, 6517-6525. [PubMed Abstract]
Three-Repeat Human Telomere Forms (3+1) G-Quadruplex in Na+ solution
We have recently solved the NMR solution structure of a DNA fragment of the human telomere that contains three guanine tracts. The d(GGGTTAGGGTTAGGGT) sequence forms a unique asymmetric dimeric quadruplex, in which the core consisting of three stacked G-tetrads, involves all three G-tracts of one strand and only the 3'-end G-tract of the other strand. In this (3+1) quadruplex assembly, three G-tracts are oriented in one direction and the fourth is oriented in the opposite direction. This new folding topology may have implications for models of t-loop formation, and for ligand design targeted to telomeric G-quadruplex folds.
Zhang, N., Phan, A. T. & Patel, D. J. (2005). (3 + 1) assembly of three human telomeric repeats into an asymmetric dimeric G-quadruplex. J. Am. Chem. Soc. 127, 17277-17285. [PubMed Abstract]
G-Quadruplexes Formed by Oncogenic Promotor Sequences
c-kit Oncogenic Promotor Sequnece
The c-kit oncogene is an important target in the treatment of gastrointestinal tumors. Our collaborator Dr. Stephen Neidle at the University of London has suggested that a potential approach to inhibition of the expression of this gene involves selective stabilization of G-quadruplex structures that may be induced to form in the c-kit promotor region. We have solved the solution structure of an unprecedented intermolecular G-quadruplex formed by a G-rich sequence in the c-kit promotor in K+ solution. The structure represents a new folding topology with several unique features. Most strikingly, an isolated guanine is involved in G-tetrad core formation, despite the presence of four three-guanine tracts. There are four loops: two single-residue double-chain-reversal loops, a two-residue loop, and a five-residue stem-loop, which contain base-pairing alignments. This unique structural scaffold provides a highly specific platform for the future design of ligands specifically targeted to the promotor DNA of c-kit.
Phan, A. T., Kuryavyi, V., Burge, S., Neidle, S. & Patel, D. J. (2007). Structure of an unprecedented G-quadruplex scaffold adopted by the human c-kit promotor. J. Am. Chem. Soc. 129, 4386-4392. [PubMed Abstract]
c-myc Oncogenic Promotor Sequences
Human c-myc is a transcription factor that is central to regulation of cell growth, proliferation, differentiation and apoptosis. The c-myc gene that encodes this protein is tightly regulated in normal cells and its aberrant overexpression is associated with the progression of many cancers. An important element in the c-myc promotor region, termed nuclease hypersensitivity element III1, controls the majority of c-myc transcription. In particular, the Laurence Hurley laboratory at the University of Arizona was the first to establish that the purine-rich strand of this element, which contains six guanine tracts, is capable of G-quadruplex formation involving stacked G.G.G.G tetrads. We have recently solved the NMR solution structure of a DNA fragment containing five guanine tracts from this region. The G-quadruplex fold is unusual in that it contains a core of three stacked guanine tetrads formed by four parallel guanine tracts with all anti guanines and a snapback 3'-end syn guanine. We have also solved the structure of the complex between this G-quadruplex and the cationic porphyrin TMPyP4. This structural information, combined with details of small molecule interaction, provides a platform for the design of anticancer drugs targeting multi-guanine tract sequences that are found in the c-myc and other oncogenic promoters.
Phan, A. T., Kuryavyi, V., Gaw, H. Y. & Patel, D. J. (2005). Targeting anticancer drugs to a parallel-stranded snapback G-quadruplex formed by five-guanine tracts of the human c-myc promotor. Nat. Chem. Biol. 1, 167-173. [PubMed Abstract]
Higher Order G-Quadruplex Scaffolds
G-Quadruplex Inhibitors of HIV-1 Integrase
HIV-1 integrase catalyzes the integration of proviral DNA into the host-cell genome, a reaction critical for efficient viral replication. Guanine-rich oligonucleotides have been identified as potent inhibitors of HIV-1 integrase. In particular, the laboratory of our collaborator Simon Litvak in Bordeaux has identified a 16-nt guanine-rich d(G4TG3AG2AG3T) sequence, designated 93del, which inhibits both the processing and strand transfer functions of HIV-1 integrase at nanomolar concentrations. NMR studies establish that the 93del sequence adopts an unusually stable dimeric quadruplex architecture in K solution. Within each 16-nt monomer subunit, an A.(G.G.G.G) pentad is sandwiched between two G.G.G.G tetrads, all G-stretches are parallel, and all guanines are anti with the exception of G1, which is syn. Dimer formation is achieved through mutual pairing of G1 of one monomer and three guanines of the other monomer, to complete G-tetrad formation. Results of NMR and of integrase inhibition assays on loop-modified sequences have provided insights into the potential design of improved HIV-1 integrase inhibitors. A model, based on molecular docking approaches, has also been proposed for positioning the 93del dimeric DNA quadruplex within a basic channel/canyon formed between subunits of the dimer of dimer alignment of HIV-1 integrase.
Phan, A. T., Kuryavyi, V., Ma, J. B., Andreola, M. L. & Patel, D. J. (2005). An interlocked dimeric parallel-stranded DNA quadruplex: a potent inhibitor of HIV-1 integrase. Proc. Natl. Acad. Scis. USA 102, 634-639. [PubMed Abstract]