RESUMO
Guanine-rich nucleic acids can form intramolecularly folded four-stranded structures known as G-quadruplexes (G4s). Traditionally, G4 research has focused on short, highly modified DNA or RNA sequences that form well-defined homogeneous compact structures. However, the existence of longer sequences with multiple G4 repeats, from proto-oncogene promoters to telomeres, suggests the potential for more complex higher-order structures with multiple G4 units that might offer selective drug-targeting sites for therapeutic development. These larger structures present significant challenges for structural characterization by traditional high-resolution methods like multi-dimensional NMR and X-ray crystallography due to their molecular complexity. To address this current challenge, we have developed an integrated structural biology (ISB) platform, combining experimental and computational methods to determine self-consistent molecular models of higher-order G4s (xG4s). Here we outline our ISB method using two recent examples from our lab, an extended c-Myc promoter and long human telomere G4 repeats, that highlights the utility and generality of our approach to characterizing biologically relevant xG4s.
RESUMO
Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are transcriptional regulators. Little structural information exists for these G4s embedded within duplexes, their presumed genomic environment. Here, we report the 7.4 Å resolution structure and dynamics of a 28.5 kDa duplex-G4-duplex (DGD) model system using cryo-EM, molecular dynamics, and small-angle X-ray scattering (SAXS) studies. The DGD cryo-EM refined model features a 53° bend induced by a stacked duplex-G4 interaction at the 5' G-tetrad interface with a persistently unstacked 3' duplex. The surrogate complement poly dT loop preferably stacks onto the 3' G-tetrad interface resulting in occlusion of both 5' and 3' tetrad interfaces. Structural analysis shows that the DGD model is quantifiably more druggable than the monomeric G4 structure alone and represents a new structural drug target. Our results illustrate how the integration of cryo-EM, MD, and SAXS can reveal complementary detailed static and dynamic structural information on DNA G4 systems.
Assuntos
Quadruplex G , Espalhamento a Baixo Ângulo , Microscopia Crioeletrônica , Difração de Raios X , DNA/químicaRESUMO
We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned. We report here five structures of long (34-70 nt) wild-type sequences selected from three cancer-related promoters: c-Myc, c-Kit and k-Ras. Each sequence studied has a unique structure. Three sequences form structures with two contiguous, stacked, G-quadruplex units. One longer sequence from c-Myc forms a structure with three contiguous stacked quadruplexes. A longer c-Kit sequence forms a quadruplex-hairpin structure. Each structure exhibits interfacial regions between stacked quadruplexes or novel loop geometries that are possible druggable targets. We also report methodological advances in our integrated structural biology approach, which now includes quantitative CD for counting stacked G-tetrads, DNaseI cleavage for hairpin detection and SAXS model refinement. Our results suggest that higher-order quadruplex assemblies may be a common feature within the genome, rather than simple single quadruplex structures.
Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Dicroísmo Circular , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
G-quadruplexes (G4s) are distinctive four-stranded DNA or RNA structures found within cells that are thought to play functional roles in gene regulation and transcription, translation, recombination, and DNA damage/repair. While G4 structures can be uni-, bi-, or tetramolecular with respect to strands, folded unimolecular conformations are most significant in vivo. Unimolecular G4 can potentially form in sequences with runs of guanines interspersed with what will become loops in the folded structure: 5'GxLyGxLyGxLyGx, where x is typically 2-4 and y is highly variable. Such sequences are highly conserved and specifically located in genomes. In the folded structure, guanines from each run combine to form planar tetrads with four hydrogen-bonded guanine bases; these tetrads stack on one another to produce four strand segments aligned in specific parallel or antiparallel orientations, connected by the loop sequences. Three types of loops (lateral, diagonal, or "propeller") have been identified. The stacked tetrads form a central cavity that features strong coordination sites for monovalent cations that stabilize the G4 structure, with potassium or sodium preferred. A single monomeric G4 typically forms from a sequence containing roughly 20-30 nucleotides. Such short sequences have been the primary focus of X-ray crystallographic or NMR studies that have produced high-resolution structures of a variety of monomeric G4 conformations. These structures are often used as the basis for drug design efforts to modulate G4 function.We believe that the focus on monomeric G4 structures formed by such short sequences is perhaps myopic. Such short sequences for structural studies are often arbitrarily selected and removed from their native genomic sequence context, and then are often changed from their native sequences by base substitutions or deletions intended to optimize the formation of a homogeneous G4 conformation. We believe instead that G-quadruplexes prefer company and that in a longer natural sequence context multiple adjacent G4 units can form to combine into more complex multimeric G4 structures with richer topographies than simple monomeric forms. Bioinformatic searches of the human genome show that longer sequences with the potential for forming multiple G4 units are common. Telomeric DNA, for example, has a single-stranded overhang of hundreds of nucleotides with the requisite repetitive sequence with the potential for formation of multiple G4s. Numerous extended promoter sequences have similar potentials for multimeric G4 formation. X-ray crystallography and NMR methods are challenged by these longer sequences (>30 nt), so other tools are needed to explore the possible multimeric G4 landscape. We have implemented an integrated structural biology approach to address this challenge. This approach integrates experimental biophysical results with atomic-level molecular modeling and molecular dynamics simulations that provide quantitatively testable model structures. In every long sequence we have studied so far, we found that multimeric G4 structures readily form, with a surprising diversity of structures dependent on the exact native sequence used. In some cases, stable hairpin duplexes form along with G4 units to provide an even richer landscape. This Account provides an overview of our approach and recent progress and provides a new perspective on the G-quadruplex folding landscape.
Assuntos
Quadruplex G , Humanos , DNA/química , Telômero , Guanina/química , Simulação de Dinâmica Molecular , Nucleotídeos , Conformação de Ácido NucleicoRESUMO
Human telomeres contain the repeat DNA sequence 5'-d(TTAGGG), with duplex regions that are several kilobases long terminating in a 3' single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate models proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5'-d(TTAGGG)n, with n = 8, 12 and 16, fold into multimeric structures containing the maximal number (2, 3 and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of â¼34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.
Assuntos
Quadruplex G , Telômero/química , Dicroísmo Circular , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (-10.1 ± 0.3 kcal mol-1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.
Assuntos
Quadruplex G , Proteínas de Ligação a Telômeros/metabolismo , Telômero/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Complexo Shelterina , Proteínas de Ligação a Telômeros/química , TermodinâmicaRESUMO
The structure of the 68 nt sequence with G-quadruplex forming potential within the hTERT promoter is disputed. One model features a structure with three stacked parallel G-quadruplex units, while another features an unusual duplex hairpin structure adjoined to two stacked parallel and antiparallel quadruplexes. We report here the results of an integrated structural biology study designed to distinguish between these possibilities. As part of our study, we designed a sequence with an optimized hairpin structure and show that its biophysical and biochemical properties are inconsistent with the structure formed by the hTERT wild-type sequence. By using circular dichroism, thermal denaturation, nuclear magnetic resonance spectroscopy, analytical ultracentrifugation, small-angle X-ray scattering, molecular dynamics simulations and a DNase I cleavage assay we found that the wild type hTERT core promoter folds into a stacked, three-parallel G-quadruplex structure. The hairpin structure is inconsistent with all of our experimental data obtained with the wild-type sequence. All-atom models for both structures were constructed using molecular dynamics simulations. These models accurately predicted the experimental hydrodynamic properties measured for each structure. We found with certainty that the wild-type hTERT promoter sequence does not form a hairpin structure in solution, but rather folds into a compact stacked three-G-quadruplex conformation.
Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Telomerase/genética , Sequência de Bases , Dicroísmo Circular , DNA/química , Humanos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
G-quadruplexes (G4s) are four-stranded nucleic acid secondary structures that form within guanine-rich regions of chromatin. G4 motifs are abundant in the genome, with a sizable proportion (â¼40%) existing within gene promoter regions. G4s are proven epigenetic features that decorate the promoter landscape as binding centers for transcription factors. Stabilizing or disrupting promoter G4s can directly influence adjacent gene transcription, making G4s attractive as indirect drug targets for hard-to-target proteins, particularly in cancer. However, no G4 ligands have progressed through clinical trials, mostly owing to off targeting effects. A major hurdle in G4 drug discovery is the lack of distinctiveness of the small monomeric G4 structures currently used as receptors. This mini review describes and contrasts monomeric and higher-order G-quadruplex structure and function and provides a rationale for switching focus to the higher-order forms as selective molecular targets. The human telomerase reverse transcriptase (hTERT) core promoter G-quadruplex is then used as a case study that highlights the potential for higher-order G4s as selective indirect inhibitors of hard-to-target proteins in cancer.
RESUMO
DNA G-quadruplexes (G4s) are now widely accepted as viable targets in the pursuit of anticancer therapeutics. To date, few small molecules have been identified that exhibit selectivity for G4s over alternative forms of DNA, such as the ubiquitous duplex. We posit that the lack of current ligand specificity arises for multiple reasons: G4 atomic models are often small, monomeric, single quadruplex structures with few or no druggable pockets; targeting G-tetrad faces frequently results in the enrichment of extended electron-deficient polyaromatic end-pasting scaffolds; and virtual drug discovery efforts often under-sample chemical search space. We show that by addressing these issues we can enrich for non-standard molecular templates that exhibit high selectivity towards G4s over other forms of DNA. We performed an extensive virtual screen against the higher-order hTERT core promoter G4 that we have previously characterized, targeting 12 of its unique loop and groove pockets using libraries containing 40 million drug-like compounds for each screen. Using our drug discovery funnel approach, which utilizes high-throughput fluorescence thermal shift assay (FTSA) screens, microscale thermophoresis (MST), and orthogonal biophysical methods, we have identified multiple unique G4 binding scaffolds. We subsequently used two rounds of catalogue-based SAR to increase the affinity of a disubstituted 2-aminoethyl-quinazoline that stabilizes the higher-order hTERT G-quadruplex by binding across its G4 junctional sites. We show selectivity of its binding affinity towards hTERT is virtually unaffected in the presence of near-physiological levels of duplex DNA, and that this molecule downregulates hTERT transcription in breast cancer cells.
Assuntos
Quadruplex G , DNA/genética , Descoberta de Drogas , Ligantes , Regiões Promotoras GenéticasRESUMO
The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 -DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.
Assuntos
Espectrometria de Fluorescência , Proteínas de Ligação a Telômeros/metabolismo , Temperatura , Quadruplex G , Humanos , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Complexo Shelterina , Telômero/química , Telômero/metabolismo , Proteínas de Ligação a Telômeros/químicaRESUMO
Analytical ultracentrifugation is a powerful biophysical tool that provides information about G-quadruplex structure, stability, and binding reactivity. This chapter provides a simplified explanation of the method, along with examples of how it can be used to characterize G4 formation and to monitor small-molecule binding.
Assuntos
Quadruplex G , Ultracentrifugação/métodos , Peso Molecular , Relação Estrutura-AtividadeRESUMO
Over the past two decades biologists and bioinformaticians have unearthed substantial evidence supporting a role for G-quadruplexes as important mediators of biological processes. This includes telomere damage signaling, transcriptional activity, and splicing. Both their structural heterogeneity and their abundance in oncogene promoters makes them ideal targets for drug discovery. Currently, there are hundreds of deposited DNA and RNA quadruplex atomic structures which have allowed researchers to begin using in silico drug screening approaches to develop novel stabilizing ligands. Here we provide a review of the past decade of G-quadruplex virtual drug discovery approaches and campaigns. With this we introduce relevant virtual screening platforms followed by a discussion of best practices to assist future G4 VS campaigns.