Guanine Quadruplex Research Articles

Ruthenation of Non-stacked Guanines in DNA G-Quadruplex Structures: Enhancement of c-MYC Expression.

Guanine quadruplexes (GQs) are compact four‐stranded DNA structures that play a key role in the control of a variety of biological processes, including gene transcription. Bulky ruthenium complexes featuring a bipyridine, a terpyridine, and one exchangeable ligand ([Ru(terpy)(bpy)X]n+) are able to metalate exposed guanines present in the GQ of the c‐MYC promoter region that are not involved in quadruplex base pairing. qRT‐PCR and western‐blot experiments indicated that the complexes promote a remarkable increase in the expression of this oncogene. We also show that exchangeable thioether ligands (X=RSR′, Met) allow regulation of the metalating activity of the complex with visible light.

Development of Antithrombotic Aptamers: From Recognizing Elements to Drugs.

Blood hemostasis is attained with two sophisticated interconnected network systems, a coagulation cascade and a platelet activation system. Multiple inhibitors were developed to various components of both systems to prevent thrombosis-related morbid events that are of extremely high frequency in the human population. Antithrombotic inhibitors possess both positive and negative aspects. One of the essential modern requirements is a controllable mode of action for both anticoagulants and antiplatelets that could be achieved due to the high affinity and specificity of the inhibitor, as well as a possibility to apply an antidote, which quickly annihilates activity of the inhibitor and restores the proper hemostasis. Aptamers are DNA or RNA oligonucleotides with particular tertiary structure, such as DNA guanine quadruplex. Besides antibodies and other peptides/proteins, aptamers are one more example of the molecular recognizing elements that specifically bind to the target. Therefore, aptamers could be developed into a promising novel class of the drugs with high affinity, specificity, innate low toxicity, and rational antidote. Several aptamers with prospective antithrombotic activity have been reviewed; some of them are in preclinical and clinical trials.

Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells.

We have previously shown that clusters of guanine quadruplex (G4) structures can form in the human herpes simplex-1 (HSV-1) genome. Here we used immunofluorescence and immune-electron microscopy with a G4-specific monoclonal antibody to visualize G4 structures in HSV-1 infected cells. We found that G4 formation and localization within the cells was virus cycle dependent: viral G4s peaked at the time of viral DNA replication in the cell nucleus, moved to the nuclear membrane at the time of virus nuclear egress and were later found in HSV-1 immature virions released from the cell nucleus. Colocalization of G4s with ICP8, a viral DNA processing protein, was observed in viral replication compartments. G4s were lost upon treatment with DNAse and inhibitors of HSV-1 DNA replication. The notable increase in G4s upon HSV-1 infection suggests a key role of these structures in the HSV-1 biology and indicates new targets to control both the lytic and latent infection.

P53 binds human telomeric G-quadruplex invitro.

The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.

Steady-State and Time-Resolved Studies into the Origin of the Intrinsic Fluorescence of G-Quadruplexes.

Stretches of guanines in DNA and RNA can fold into guanine quadruplex structures (GQSs). These structures protect telomeres in DNA and regulate gene expression in RNA. GQSs have an intrinsic fluorescence that is sensitive to different parameters, including loop sequence and length. However, the dependence of GQS fluorescence on solution and sequence parameters and the origin of this fluorescence are poorly understood. Herein we examine effects of dangling nucleotides and cosolute conditions on GQS fluorescence using both steady-state and time-resolved fluorescence spectroscopy. The quantum yield of dGGGTGGGTGGGTGGG, termed "dG3T", is found to be modest at ∼2 × 10(-3). Nevertheless, dG3T and its variants are significantly brighter than the common nucleic acid fluorophore 2-aminopurine (2AP) largely due to their sizable extinction coefficients. Dangling 5'-end nucleotides generally reduce emission and blue-shift the resultant spectrum, whereas dangling 3'-end nucleotides slightly enhance fluorescence, particularly on the red side of the emission band. Time-resolved fluorescence decays are broadly distributed in time and require three exponential components for accurate fits. Time-resolved emission spectra suggest the presence of two emitting populations centered at ∼330 and ∼390 nm, with the redder component being a well-defined long-lived (∼1 ns) entity. Insights into GQS fluorescence obtained here should be useful in designing brighter intrinsic RNA and DNA quadruplexes for use in label-free biotechnological applications.

QuadBase2: web server for multiplexed guanine quadruplex mining and visualization.

DNA guanine quadruplexes or G4s are non-canonical DNA secondary structures which affect genomic processes like replication, transcription and recombination. G4s are computationally identified by specific nucleotide motifs which are also called putative G4 (PG4) motifs. Despite the general relevance of these structures, there is currently no tool available that can allow batch queries and genome-wide analysis of these motifs in a user-friendly interface. QuadBase2 (quadbase.igib.res.in) presents a completely reinvented web server version of previously published QuadBase database. QuadBase2 enables users to mine PG4 motifs in up to 178 eukaryotes through the EuQuad module. This module interfaces with Ensembl Compara database, to allow users mine PG4 motifs in the orthologues of genes of interest across eukaryotes. PG4 motifs can be mined across genes and their promoter sequences in 1719 prokaryotes through ProQuad module. This module includes a feature that allows genome-wide mining of PG4 motifs and their visualization as circular histograms. TetraplexFinder, the module for mining PG4 motifs in user-provided sequences is now capable of handling up to 20 MB of data. QuadBase2 is a comprehensive PG4 motif mining tool that further expands the configurations and algorithms for mining PG4 motifs in a user-friendly way.

G-register exchange dynamics in guanine quadruplexes.

G-quadruplexes (GQs) are 4-stranded DNA structures formed by tracts of stacked, Hoogsteen-hydrogen bonded guanosines. GQs are found in gene promoters and telomeres where they regulate gene transcription and telomere elongation. Though GQ structures are well-characterized, many aspects of their conformational dynamics are poorly understood. For example, when there are surplus guanosines in some of the tracts, they can slide with respect to one another, a process we term G-register (GR) exchange. These motions could in principle entropically stabilize the folded state, crucially benefitting GQs as their stabilities are closely tied to biological function. We have developed a method for characterizing GR exchange where each isomer in the wild-type conformational ensemble is trapped by mutation and thermal denaturation data for the set of trapped mutants and wild-type are analyzed simultaneously. This yields GR isomer populations as a function of temperature, quantifies conformational entropy and sheds light on correlated sliding motions of the G-tracts. We measured entropic stabilizations from GR exchange up to 14.3 ± 1.6 J mol−1 K−1, with melting temperature increases up to 7.3 ± 1.6°C. Furthermore, bioinformatic analysis suggests a majority of putative human GQ sequences are capable of GR exchange, pointing to the generality of this phenomenon.

Circular Dichroism of DNA G-Quadruplexes: Combining Modeling and Spectroscopy To Unravel Complex Structures.

We report on the comparison between the computational and experimental determination of electronic circular dichroism spectra of different guanine quadruplexes obtained from human telomeric sequences. In particular the difference between parallel, antiparallel, and hybrid structures is evidenced, as well as the induction of transitions between the polymorphs depending on the solution environment. Extensive molecular dynamics simulations (MD) are used to probe the conformational space of the different quadruplexes, and subsequently state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM) techniques coupled with excitonic semiempirical Hamiltonian are used to simulate the macromolecular induced circular dichroism. The coupling of spectroscopy and molecular simulation allows an efficient one-to-one mapping between structures and optical properties, offering a way to disentangle the rich, yet complicated, quantity of information embedded in circular dichroism spectra. We show that our methodology is robust and efficient and allows us to take into account subtle conformational changes. As such, it could be used as an efficient tool to investigate structural modification upon DNA/drug interactions.

Can We Execute Reliable MM-PBSA Free Energy Computations of Relative Stabilities of Different Guanine Quadruplex Folds?

The self-assembly and stability of DNA G-quadruplexes (GQs) are affected by the intrinsic stability of different GpG base steps embedded in their G-quartet stems. We have carried out MD simulations followed by MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) free energy calculations on all the experimentally observed three-quartet intramolecular human telomeric GQ topologies. We also studied antiparallel GQ models with alternative syn-anti patterns of the G-quartets. We tested different ions, dihedral variants of the DNA force field, water models, and simulation lengths. In total, ∼35 μs of simulations have been carried out. The systems studied here are considerably more complete than the previously analyzed two-quartet stems. Among other effects, our computations included the stem-loop coupling and ion-ion interactions inside the stem. The calculations showed a broad agreement with the earlier predictions. However, the increase in the completeness of the system was associated with increased noise of the free energy data which could be related, for example, to the presence of long-lived loop substates and rather complex dynamics for the two bound ions inside the G-stem. As a result, the MM-PBSA data were noisy and we could not improve their quantitative convergence even by expanding the simulations to 2.5 μs long trajectories. We also suggest that the quality of MM-based free energy computations based on MD simulations of complete GQs is more sensitive to the neglect of explicit polarization effects, which, in real systems, are associated with the presence of multiple closely spaced ions inside the GQs. Thus, although the MM-PBSA procedure provides very useful insights that complement the structural-dynamics data from MD trajectories of GQs, the method is far from reaching quantitative accuracy. Our conclusions are in agreement with critical assessments of the MM-PBSA methodology available in contemporary literature for other types of problems.

Derivation of Reliable Geometries in QM Calculations of DNA Structures: Explicit Solvent QM/MM and Restrained Implicit Solvent QM Optimizations of G-Quadruplexes

Modern dispersion-corrected DFT methods have made it possible to perform reliable QM studies on complete nucleic acid (NA) building blocks having hundreds of atoms. Such calculations, although still limited to investigations of potential energy surfaces, enhance the portfolio of computational methods applicable to NAs and offer considerably more accurate intrinsic descriptions of NAs than standard MM. However, in practice such calculations are hampered by the use of implicit solvent environments and truncation of the systems. Conventional QM optimizations are spoiled by spurious intramolecular interactions and severe structural deformations. Here we compare two approaches designed to suppress such artifacts: partially restrained continuum solvent QM and explicit solvent QM/MM optimizations. We report geometry relaxations of a set of diverse double-quartet guanine quadruplex (GQ) DNA stems. Both methods provide neat structures without major artifacts. However, each one also has distinct weaknesses. In restrained optimizations, all errors in the target geometries (i.e., low-resolution X-ray and NMR structures) are transferred to the optimized geometries. In QM/MM, the initial solvent configuration causes some heterogeneity in the geometries. Nevertheless, both approaches represent a decisive step forward compared to conventional optimizations. We refine earlier computations that revealed sizable differences in the relative energies of GQ stems computed with AMBER MM and QM. We also explore the dependence of the QM/MM results on the applied computational protocol.

Distance measurements between paramagnetic ligands bound to parallel stranded guanine quadruplexes.

Aside from a double helix, deoxyribonucleic acid (DNA) folds into non-canonical structures, one of which is the guanine quadruplex. Cationic porphyrins bind guanine quadruplexes, but the effects of ligand binding on the structure of guanine quadruplexes with more than four contiguous guanine quartets remains to be fully elucidated. Double electron-electron resonance (DEER) spectroscopy conducted at 9.5 GHz (X-band) using broadband, shaped inversion pulses was used to measure the distances between cationic copper porphyrins bound to model parallel-stranded guanine quadruplexes with increasing numbers of guanine quartets. A single Gaussian component was found to best model the time domain datasets, characteristic of a 2 : 1 binding stoichiometry between the porphyrins and each quadruplex. The measured Cu(2+)-Cu(2+) distances were found to be linearly proportional with the number of guanines. Rather unexpectedly, the ligand end-stacking distance was found to monotonically decreases the overall quadruplex length was extended, suggesting a conformational change in the quadruplex secondary structure dependent upon the number of successive guanine quartets.

Exploring the characterization tools of Guanine-Quadruplexes.

Occurrence of guanine-rich sequences throughout the genome at specific locations like chromosomal ends (telomeres), promoters and Untranslated regions (UTR's) is very well documented. Quite recently, visualization of guanine-quadruplex in human and mammalian cells have also provided a very significant evidence for the in vivo existence of guanine-quadruplex, reconfirming their biological relevance in cellular processes like replication, transcription, recombination, etc. Guanine quadruplexes have enormous potential of exhibiting various topologies which differ, by number/ orientation of strands or loop orientations etc. Some relatively new polymorphic structures like 3+1 quadruplex, G-triplex, and Tri-G-quadruplex have also been proposed for the guanine-rich sequences. Various biochemical and biophysical techniques have been used to characterize these multistranded DNA structures. An extensive review of the mechanistic models of the already existing and newly emerging techniques is actually required, which may further facilitate our understanding about these structures. This review aims to summarize some of these techniques along with their requirements and limitations, which might further give some insights for the fine tuning of the solution and environmental conditions needed for facilitating guanine-quadruplex formation.

An ion-controlled four-color fluorescent telomeric switch on DNA origami structures.

The folding of single-stranded telomeric DNA into guanine (G) quadruplexes is a conformational change that plays a major role in sensing and drug targeting. The telomeric DNA can be placed on DNA origami nanostructures to make the folding process extremely selective for K(+) ions even in the presence of high Na(+) concentrations. Here, we demonstrate that the K(+)-selective G-quadruplex formation is reversible when using a cryptand to remove K(+) from the G-quadruplex. We present a full characterization of the reversible switching between single-stranded telomeric DNA and G-quadruplex structures using Förster resonance energy transfer (FRET) between the dyes fluorescein (FAM) and cyanine3 (Cy3). When attached to the DNA origami platform, the G-quadruplex switch can be incorporated into more complex photonic networks, which is demonstrated for a three-color and a four-color FRET cascade from FAM over Cy3 and Cy5 to IRDye700 with G-quadruplex-Cy3 acting as a switchable transmitter.

The role of alkali metal cations in the stabilization of guanine quadruplexes: why K(+) is the best.

The alkali metal ion affinity of guanine quadruplexes has been studied using dispersion-corrected density functional theory (DFT-D). We have done computational investigations in aqueous solution that mimics artificial supramolecular conditions where guanine bases assemble into stacked quartets as well as biological environments in which telomeric quadruplexes are formed. In both cases, an alkali metal cation is needed to assist self-assembly. Our quantum chemical computations on these supramolecular systems are able to reproduce the experimental order of affinity of the guanine quadruplexes for the cations Li(+), Na(+), K(+), Rb(+), and Cs(+). The strongest binding is computed between the potassium cation and the quadruplex as it occurs in nature. The desolvation and the size of alkali metal cations are thought to be responsible for the order of affinity. Until now, the relative importance of these two factors has remained unclear and debated. By assessing the quantum chemical 'size' of the cation, determining the amount of deformation of the quadruplex needed to accommodate the cation and through the energy decomposition analysis (EDA) of the interaction energy between the cation and the guanines, we reveal that the desolvation and size of the alkali metal cation are both almost equally responsible for the order of affinity.

Nanoestructuras inusuales de ácidos nucleicos basados en ADN G-cuádruple

Se reporta la preparación de un nano-ensamble basado en estructuras de ADN no canónico. Para la nanofabricación de una estructura basada en ADN el auto ensamblaje de ADN G-cuádruple (apareamiento de bases tipo hoogsteen) y ADN de doble cadena (apareamiento de bases tipo Watson-Crick) fueron utilizados. En general, un número importante de nanoestructuras se han construido explotando la capacidad de apareamiento de bases del ADN canónico de doble cadena. Hay formas alternativas para construcción utilizando otros elementos de ADN tales como G-cuadruple, motivos I, o ADN de triple cadena. Como prueba de prinicipio, previamente hemos reportado el uso de ADN de doble cadena (oligonucleótidos de ADN sintéticos cortos) con secciones de sitios no apareados capaz de mediar la formación de secciones tetramoleculares (pruebas G-cuádruple) con la finalidad de ensamblar los componentes en estructuras de alto peso molecular. Gel electroforesis como también microscopia de fuerza atómica muestran la formación de nanofibras. La electroforesis de Gel como el dicroismo circular dan evidencia de la presencia de secciones G-cuádruple. De las imágenes de microscopía de fuerza atómica se estimó que el largo de las estructuras va de 250 a 2000 nm con altitud de 0.45 a 4.0 nm. Aquí presentamos otro ejemplo de tales nanofibras. Sugerimos que metodologías similares pueden ser usa-das para construir nanoestructuras más complejas que saquen provecho de las propiedades de distintos nano-rarezas de ADN para aplicaciones capaces de realizar tareas útiles.

Amyloid Precursor Protein Translation Is Regulated by a 3'UTR Guanine Quadruplex.

A central event in Alzheimer’s disease is the accumulation of amyloid β (Aβ) peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP). APP overexpression leads to increased Aβ generation and Alzheimer’s disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aβ levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aβ levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes), non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3’UTR (untranslated region) at residues 3008–3027 (NM_201414.2). This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3’UTR G-quadruplex as a novel mechanism regulating APP expression.

Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro.

Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase Ib of D. radiodurans (DraTopoIB) could change the electrophoretic mobility of fast migrating intramolecular recF-G4 DNA into the slow migrating species. DraTopoIB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular recF-G4 DNA structures stabilized by K+. On the contrary, when DraTopoIB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopoIB. This implies that DraTopoIB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopoIB activity on intramolecular G4 DNA structures. These results suggested that DraTopoIB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

Refinement of the Sugar-Phosphate Backbone Torsion Beta for AMBER Force Fields Improves the Description of Z- and B-DNA.

Z-DNA duplexes are a particularly complicated test case for current force fields. We performed a set of explicit solvent molecular dynamics (MD) simulations with various AMBER force field parametrizations including our recent refinements of the ε/ζ and glycosidic torsions. None of these force fields described the ZI/ZII and other backbone substates correctly, and all of them underpredicted the population of the important ZI substate. We show that this underprediction can be attributed to an inaccurate potential for the sugar-phosphate backbone torsion angle β. We suggest a refinement of this potential, β(OL1), which was derived using our recently introduced methodology that includes conformation-dependent solvation effects. The new potential significantly increases the stability of the dominant ZI backbone substate and improves the overall description of the Z-DNA backbone. It also has a positive (albeit small) impact on another important DNA form, the antiparallel guanine quadruplex (G-DNA), and improves the description of the canonical B-DNA backbone by increasing the population of BII backbone substates, providing a better agreement with experiment. We recommend using β(OL1) in combination with our previously introduced corrections, εζ(OL1) and χ(OL4), (the combination being named OL15) as a possible alternative to the current β torsion potential for more accurate modeling of nucleic acids.

A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy.

AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

Hairpins participating in folding of human telomeric sequence quadruplexes studied by standard and T-REMD simulations.

DNA G-hairpins are potential key structures participating in folding of human telomeric guanine quadruplexes (GQ). We examined their properties by standard MD simulations starting from the folded state and long T-REMD starting from the unfolded state, accumulating ∼130 μs of atomistic simulations. Antiparallel G-hairpins should spontaneously form in all stages of the folding to support lateral and diagonal loops, with sub-μs scale rearrangements between them. We found no clear predisposition for direct folding into specific GQ topologies with specific syn/anti patterns. Our key prediction stemming from the T-REMD is that an ideal unfolded ensemble of the full GQ sequence populates all 4096 syn/anti combinations of its four G-stretches. The simulations can propose idealized folding pathways but we explain that such few-state pathways may be misleading. In the context of the available experimental data, the simulations strongly suggest that the GQ folding could be best understood by the kinetic partitioning mechanism with a set of deep competing minima on the folding landscape, with only a small fraction of molecules directly folding to the native fold. The landscape should further include non-specific collapse processes where the molecules move via diffusion and consecutive random rare transitions, which could, e.g. structure the propeller loops.