Dinshaw Patel: Higher Order G-Quadruplex Architectures

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. Several 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 pilE G4 Sequence

Neisseria gonorrhoeae is an obligate human pathogen that can escape immune surveillance through antigenic variation of surface structures such as pili. A G-quadruplex-forming (G4) sequence (5´-G3TG3TTG3TG3) located upstream of the N. gonorrhoeae pilin expression locus (pilE) is necessary for initiation of pilin antigenic variation, a recombination-based, high-frequency, diversity-generation system.

5’-End-Stacked Dimeric Parallel G-Quadruplexes

5’-End-Stacked Dimeric Parallel G-Quadruplexes

We have determined NMR-based structures of the all-parallel-stranded monomeric and novel 5´-end-stacked dimeric pilE G-quadruplexes in monovalent cation-containing solution. We demonstrate that the three-layered all-parallel-stranded monomeric pilE G-quadruplex containing single residue double-chain-reversal loops that can be modeled without steric clashes into the three-nucleotide DNA-binding site of RecA, binds and promotes E. coli RecA mediated strand exchange in vitro. We discuss how interactions between RecA and monomeric pilE G-quadruplex could facilitate the specialized recombination reactions leading to pilin diversification.

Kuryavyi, V., Cahoon, L. A., Seifert, H. S. & Patel, D. J. (2013). RecA-binding pilE G4 sequence essential for pilin antigenic variation forms parallel-stranded monomeric and 5’-end stacked dimeric G-quadruplexes. Structure 20, 2090-2102.

G-Quadruplexes Formed by Intronic Sequences

The recent demonstration of guanine-rich repeats such as d(G3-N-G4-N2-G4-N-G3-N) with the potential for formation of polymorphic G-quadruplex scaffolds within the first intron of human genes has highlighted the need to elucidate the diversity of G-quadruplex folding topologies adopted by human intronic guanine-rich repeats.

Four-Repeat Human  Intronic G-Quadruplex in K+ Solution

Four-Repeat Human  Intronic G-Quadruplex in K+ Solution

We report on the solution structure of an unprecedented intramolecular G-quadruplex formed by the guanine-rich human chl1 intronic d(G3-N-G4-N2-G4-N-G3-N) 19-mer sequence in K+-containing solution. This G-quadruplex, composed of three stacked G-tetrads containing four syn guanines, represents a new folding topology with two unique conformational features. The first guanine is positioned within the central G-tetrad, in contrast to all previous structures of unimolecular G-quadruplexes, where the first guanine is part of the outermost G-tetrad. In addition, a V-shaped loop, spanning three G-tetrad planes, contains no bridging nucleotides. Finally, the chl1 intronic DNA G-quadruplex scaffold contains a guanine base intercalated between an extended G-G step, a feature observed in common with the catalytic site of group I introns. This unique structural scaffold provides a highly specific platform for the future design of ligands specifically targeted to intronic G-quadruplex platforms.

Kuryavyi, V. & Patel, D. J. (2010). Solution structure of an unique G-quadruplex scaffold adopted by a guanosine-rich human intronic sequence. Structure 18, 73-82.

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

Four-Repeat Human Telomere forms (3+1) G-Quadruplex in K+ solution

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 Promoter Sequences

Previous structural studies have demonstrated that nuclease hypersensitivity regions of several proto-oncogenic DNA promoters, situated upstream of transcription start sites, contain guanine-rich tracts that form intramolecular G-quadruplexes stabilized by stacked G-tetrads in monovalent cation solution. The human c-kit oncogenic promoter, an important target in the treatment of gastrointestinal tumors, contains two such stretches of guanine-rich tracts, designated c-kit1 and c-kit2. We set out to define the G-quadruplexes adopted by the guanine rich c-kit1 and c-kit2 DNA sequences.

c-kit2 Promoter Sequence

c-kit2 Promoter Sequence

Our previous NMR-based studies reported on the novel G-quadruplex scaffold of the c-kit1 promoter in K+-containing solution, where we showed for the first time that even an isolated guanine was involved in G-tetrad formation. These NMR-based studies are now extended to the c-kit2 promoter, which adopts two distinct all-parallel-stranded conformations in slow exchange, one which forms a monomeric G-quadruplex (form-I) in 200 mM K+-containing solution and the other a novel dimeric G-quadruplex (form-II) in 100 mM K+-containing solution. The c-kit2 promoter dimeric form-II G-quadruplex adopts an unprecedented all-parallel stranded topology where individual c-kit2 promoter strands span a pair of three-G-tetrad layer containing all-parallel-stranded G-quadruplexes aligned in a 3’- to 5’-end orientation, with stacking continuity between G-quadruplexes mediated by a sandwiched A•A non-canonical pair. We propose that strand exchange during recombination events within guanine-rich segments, could potentially be mediated by a synapsis intermediate involving an intergenic parallel-stranded dimeric G-quadruplex.

Kuryavyi, V., Phan, A. T. & Patel, D. J. (2010). Solution structures of all parallel-stranded monomeric and dimeric G-quadruplex scaffolds of the human c-kit2 promoter. Nucleic Acids Res. 38, 6757-6773.

c-kit1 Oncogenic Promoter Sequnece

The c-kit oncogene is an important target in the treatment of gastrointestinal tumors. Our collaborator Dr. Stephen Neidle (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 promoter 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 promoter 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 promoter. J. Am. Chem. Soc. 129, 4386-4392. [PubMed Abstract]

c-myc Oncogenic Promoter Sequences

G-Quadruplexes formed by 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 (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 promoter. Nat. Chem. Biol. 1, 167-173. [PubMed Abstract]

Higher Order G-Quadruplex Scaffolds as Drugs

G-quadruplexes represent compact folds that have the potential to target large channels/canyons in multi-subunit proteins. One such channel/canyon is generated by the dimer of dimers of HIV-1 integrase. Indeed, a guanine-rich DNA aptamer with the capability to form a higher order fold G-quadruplex was shown to inhibit both the processing and strand transfer functions of HIV-1 integrase. A challenge was to understand the principles underlying the shape complementarity between the higher order fold of the G-quadruplex aptamer and its target channel/canyon within the multimeric state of the HIV-1 integrase.

G-Quadruplex Inhibitors of HIV-1 Integrase

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 (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]