Parallel-stranded G-quadruplex Research Articles

A Study on Biologically Active Components in <i>Morinda citrifolia</i> Leaf Extract and its Anti-cancer Effect by <i>In Vitro</i> Analysis and <i>In Silico</i> Molecular Docking Method

Nowadays, most antiviral drugs are plant-based due to their low toxicity and high resistance. Morinda citrifolia (Noni) is such a perennial shrub a popular plant based medicine due to its wide therapeutic applications. The phytochemicals present in its fruit as well as its leaf have many antiviral properties and can enhance the human immune system. Herein, we report the identification of bioactive components present in the Morinda citrifolia (Noni) ethanolic leaf extract by GC-MS analysis and their biological interaction with human parallel-stranded 7-mer g-quadruplex DNA of MCF-7 using Biovia Discovery Studio software. In vitro analysis by MTT assay of the same extract reported a living cells of 59.74% at 100 µg/ml. The GC-MS analysis of ethanolic leaf extract revealed the presence of 27 bioactive components, and out of these, six major components were interacted with cancer DNA. The molecular docking interaction identified the most active bio component Phytol, with a binding energy of -27.0796 Kcal/mol. this value is much better than the commercially available doxorubicin. This novel study will provide insight into the development of bioactive components from Morinda citrifolia leaf extract against human breast cancer with minimal side effects.

Photosensitized Generation of Singlet Oxygen (1O2) from Pyropheophorbide a Specifically Bound to All Parallel-Stranded G-Quadruplex DNAs.

Pyropheophorbide a (Pyro-a), a chlorophyll metabolite, is a potential photosensitizer for photodynamic therapy (PDT), but little is known about its interaction with the target molecules for PDT, e.g., nucleic acids. Elucidation of the interaction between photosensitizers and nucleic acids will help us understand the initial process of PDT at the molecular level and hence to develop photosensitizing agents. We found that pyro-a forms a 1:1 complex with an all parallel-stranded G-quadruplex DNA and that pyro-a in the complex exhibits a quantum yield for singlet oxygen generation, with excitation at 664 nm, higher by a factor of ∼10 than that of pyro-a in an aqueous solution. These findings provided novel insights into molecular design of pyro-a-based photosensitizers to enhance their PDT efficacy.

To probe the binding of TMPyP4 to c-MYC G-quadruplex with in water and in imidazolium-based ionic liquids using spectroscopy coupled with molecular dynamics simulations

TMPyP4 is known to stabilize c-MYC Pu22 G-quadruplexes and decrease c-MYC oncogene expression. Ionic liquids (ILs) were demonstrated to modulate G-quadruplex stability, serving as attractive alternatives in nucleic acid storage for biomedical applications. However, no high-resolution structure of TMPyP4 complexed with Pu22 has been solved. Additionally, the effect of imidazolium-based ILs on G-quadruplex structure is unknown. Finally, the effect of ILs on ligand binding to G-quadruplexes has not been investigated. Using spectroscopy coupled with molecular dynamics (MD) simulations in this study, we elucidate the detailed binding interactions and effects of ligand TMPyP4 and four imidazolium-based ILs with increasing hydrophobic chains on the structure of a 22mer c-MYC parallel-stranded DNA G-quadruplex (Pu22) and the effect of these ILs on TMPyP4 binding to Pu22. UV–Vis and CD spectroscopy revealed 1) OMIM-Cl increases the melting temperature of the G-quadruplex from ∼ 80 °C to ∼ 90 °C. 2) TMPyP4 binds G-quadruplex in 2:1 ratio, 3) the four IL decreases TMPyP4 binding to Pu22 in a cation-dependent manner. MD simulations suggest that 1) TMPyP4 primarily binds to the top G-quadruplex layer, 2) the four ILs prevent TMPyP4 ligand binding via hydrophobic and electrostatic interactions. This study supports ILs could increase the thermal stability of G-quadruplex structure.

Fluorophore-mediated Photooxidation of the Guanine Heterocycle.

Fluorescent dyes are routinely used to visualize DNA or RNA in various experiments, and some dyes also act as photosensitizers capable of catalyzing oxidation reactions. The present studies explored whether the common labeling dyes fluorescein, rhodamine, BODIPY, or cyanine3 (Cy3) can function as photosensitizers to oxidize nucleic acid polymers. Photoirradiation of each dye in the presence of the guanine (G) heterocycle, which is the most sensitive toward oxidation, identified slow rates of nucleobase oxidation in the nucleoside and DNA contexts. For all four fluorophores studied, the only product detected was spiroiminodihydantoin (Sp) suggesting the dyes functioned as Type II photosensitizers and generate singlet oxygen (1O2). The nucleoside reactions were then conducted in D2O solutions, known to increase the lifetime of 1O2, which resulted in a ~6-fold increase in the Sp yield, further supporting the classification of these dyes as Type II photosensitizers. Lastly, we inspected the pattern of G reactivity with the dyes upon photoirradiation in the context of a parallel-stranded G-quadruplex. The G nucleotides in the two exterior G-tetrads were found to be oxidation prone, providing the third line of evidence that the dyes are Type II photooxidants. The present work found that the common dyes fluorescein, rhodamine, BODIPY, or Cy3 can drive G oxidation but with a slow rate and low overall yield. This will likely not impact many experiments using dyes to study nucleic acids except for those that have long exposures with high-intensity lights, such as sequencing-by-synthesis experiments using fluorescence as the readout.

The polarization dependence of 2D IR cross-peaks distinguishes parallel-stranded and antiparallel-stranded DNA G-quadruplexes

Guanine-rich nucleic acid sequences have a tendency to form four-stranded non-canonical motifs known as G-quadruplexes. These motifs may adopt a wide range of structures characterized by size, strand orientation, guanine base conformation, and fold topology. Using three K+-bound model systems, we show that vibrational coupling between guanine C6 = O and ring modes varies between parallel-stranded and antiparallel-stranded G-quadruplexes, and that such structures can be distinguished by comparison of the polarization dependences of cross-peaks in their two-dimensional infrared (2D IR) spectra. Combined with previously defined vibrational frequency trends, this analysis reveals key features of a 30-nucleotide unimolecular variant of the Bcl-2 proximal promoter that are consistent with its reported structure. This study shows that 2D IR spectroscopy is a convenient method for analyzing G-quadruplex structures that can be applied to complex sequences where traditional high-resolution methods are limited by solubility and disorder.

Effects of Heme Electronic Structure and Local Heme Environment on Catalytic Activity of a Peroxidase-Mimicking Heme-DNAzyme.

The catalytic cycle of a peroxidase-mimicking heme-DNAzyme involves an iron(IV)oxo porphyrin π-cation radical intermediate known as compound I formed through heterolytic O-O bond cleavage of an Fe3+-bound hydroperoxo ligand (Fe-OOH) in compound 0, like that of a heme enzyme such as horseradish peroxidase (HRP). Peroxidase assaying of complexes composed of chemically modified hemes possessing various electron densities of the heme iron atom (ρFe) and parallel-stranded tetrameric G-quadruplex DNAs of oligonucleotides d(TTAGGG), d(TTAGGGT), and d(TTAGGGA) was performed to elucidate the effects of the heme electronic structure and local heme environment on the catalytic activity of the heme-DNAzyme. The study revealed that the DNAzyme activity is enhanced through an increase in the ρFe and general base catalysis of the adenine base adjacent to the heme, which are reminiscent of the "push" and "pull" mechanisms in the catalytic cycle of HRP, respectively, and that the activity of the heme-DNAzyme can be independently controlled through the heme electronic structure and local heme environment. These findings allow a deeper understanding of the structure-function relationship of the peroxidase-mimicking heme-DNAzyme.

Mechanical diversity and folding intermediates of parallel-stranded G-quadruplexes with a bulge.

A significant number of sequences in the human genome form noncanonical G-quadruplexes (G4s) with bulges or a guanine vacancy. Here, we systematically characterized the mechanical stability of parallel-stranded G4s with a one to seven nucleotides bulge at various positions. Our results show that G4-forming sequences with a bulge form multiple conformations, including fully-folded G4 with high mechanical stability (unfolding forces > 40 pN), partially-folded intermediates (unfolding forces < 40 pN). The folding probability and folded populations strongly depend on the positions and lengths of the bulge. By combining a single-molecule unfolding assay, dimethyl sulfate (DMS) footprinting, and a guanine-peptide conjugate that selectively stabilizes guanine-vacancy-bearing G-quadruplexes (GVBQs), we identified that GVBQs are the major intermediates of G4s with a bulge near the 5′ or 3′ ends. The existence of multiple structures may induce different regulatory functions in many biological processes. This study also demonstrates a new strategy for selectively stabilizing the intermediates of bulged G4s to modulate their functions.

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

A cationic copolymer as a cocatalyst for a peroxidase-mimicking heme-DNAzyme.

Heme binds to a parallel-stranded G-quadruplex DNA to form a peroxidase-mimicking heme-DNAzyme. An interpolyelectrolyte complex between the heme-DNAzyme and a cationic copolymer possessing protonated amino groups was characterized and the peroxidase activity of the complex was evaluated to elucidate the effect of the polymer on the catalytic activity of the heme-DNAzyme. We found that the catalytic activity of the heme-DNAzyme is enhanced through the formation of the interpolyelectrolyte complex due to the general acid catalysis of protonated amino groups of the polymer, enhancing the formation of the iron(IV)oxo porphyrin π-cation radical intermediate known as Compound I. This finding indicates that the polymer with protonated amino groups can act as a cocatalyst for the heme-DNAzyme in the oxidation catalysis. We also found that the enhancement of the activity of the heme-DNAzyme by the polymer depends on the local heme environment such as the negative charge density in the proximity of the heme and substrate accessibility to the heme. These findings provide novel insights as to molecular design of the heme-DNAzyme for enhancing its catalytic activity.

Stepwise binding of a cationic phthalocyanine derivative to an all parallel-stranded tetrameric G-quadruplex DNA

Interaction between an Ga(III) phthalocyanine (Pc) derivative bearing eight N-methylpyridinium groups at peripheral β-positions (2,3,6,7,10,11,14,15-octakis-[N-methyl-(4-methylpyridinium-3-yloxy)phthalocyaninato] chloro gallium(III) iodide (GaPc)) and an all parallel-stranded tetrameric G-quadruplex formed from a heptanucleotide d(TTAGGGT) ([d(TTAGGGT)]4) has been investigated to elucidate the molecular recognition of G-quadruplex DNA by the Pc derivative, which provides a useful insight as to the design of G-quadruplex ligands suitable for various in vitro and in vivo applications. We found that GaPc binds to the A3G4 and G6T7 steps of [d(TTAGGGT)]4, with binding constants of (21 ± 2) × 106 and (0.09 ± 0.06) × 106 M−1, respectively, to form a 2:1 complex. Obviously, upon the binding of GaPc to each of the sites, the π-π stacking and electrostatic interactions of the Pc moiety and positively-charged side chains of GaPc with a G-quartet and the negatively-charged phosphate groups in nearby phosphodiester bonds of the DNA, respectively, are major driving forces for the complexation. Considering the similarity in the local structural environment between the A3G4 and G6T7 steps of [d(TTAGGGT)]4, the remarkably large difference in the GaPc-binding affinity between them is most likely accounted for by the effect of the polarity of the GaPc-binding site on the intermolecular electrostatic interaction. This finding provides valuable insights as to the design of Pc derivatives as G-quadruplex ligands.

Photogeneration of Reactive Oxygen Species from Water-Soluble Phthalocyanine Derivatives Bound to a G-Quadruplex DNA

Abstract Water-soluble phthalocyanine (Pc) derivatives have been studied extensively as not only effective photosensitizers for photodynamic therapy (PDT), but also G-quadruplex ligands which have the potential to be anticancer drugs. We investigated the photogeneration of reactive oxygen species (ROS) from cationic Pc derivatives, 2,3,6,7,10,11,14,15-octakis-[N-methyl-(4-methylpyridinium-3-yloxy)phthalocyaninato] chloro gallium(III) iodide (GaPc) and zinc(II) iodide (ZnPc), in the presence of an all parallel-stranded tetrameric G-quadruplex DNA, i.e., [d(TTAGGGT)4]. We found that the photo ROS generation from GaPc is suppressed in the presence of the DNA, whereas that from ZnPc is enhanced on the addition of the DNA. The suppression of the photo ROS generation for GaPc by the DNA was found to be due to electron transfer from the ground state of the guanine base of the DNA to the photo-excited state of GaPc. This finding provides a clue as to why phototoxicity of GaPc is lower than that of ZnPc in human Hep2 cells (Fujishiro et al. J. Inorg. Biochem.2019, 192, 7). On the other hand, the enhancement of the photo ROS generation from ZnPc by the DNA is possibly due to the dissociation of its non-fluorescent aggregate through its interaction with the DNA. Thus the interaction of photosensitizers with DNA and RNA is crucial for their PDT efficacy.

Characterization of Structure and Catalytic Activity of a Complex between Heme and an All Parallel-Stranded Tetrameric G-Quadruplex Formed from DNA/RNA Chimera Sequence d(TTA)r(GGG)dT

Abstract An all-parallel tetrameric G-quadruplex formed from a DNA/RNA chimera sequence, d(TTA)r(GGG)d(T) ([Chimera(7mer)]4), and its interaction with heme have been characterized. [Chimera(7mer)]4 was found to be remarkably more stable compared with a G-quadruplex formed from its DNA counterpart d(TTAGGGT), ([DNA(7mer)]4), despite the structural similarity between them, and the increased stability of [Chimera(7mer)]4 is likely to be due to the formation of interstrand hydrogen bonds between the rG5 2′-OH and rG6 amino NH2 groups. Heme bound selectively to the 3′-terminal G-quartet (rG6 G-quartet) in the rG6dT7 step of [Chimera(7mer)]4 did not exhibit the heme orientational disorder resulting from the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C2 axis, with respect to the interacting G-quartet, although it is a general feature of heme-DNA complexes. In a complex between heme and [Chimera(7mer)]4, the predominance of one isomer in the heme orientational disorder over the other one is possibly due to contacts between the rG6 2′-OH group of the G-quartet and heme side chain ones. Peroxidase activity of heme bound to a G-quartet was found to be independent of the presence of the 2′-OH group in the ribose of the constituent units of the G-quartet.

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3′-end tetrad of a DNA G-quadruplex

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) – a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3′-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5′-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.

Why do G-quadruplexes dimerize through the 5’-ends? Driving forces for G4 DNA dimerization examined in atomic detail

G-quadruplexes (G4) are secondary structures formed by guanine-rich nucleic acid sequences and shown to exist in living cells where they participate in regulation of gene expression and chromosome maintenance. G-quadruplexes with solvent-exposed guanine tetrads show the tendency to associate together through cofacial stacking, which may be important for packaging of G4-forming sequences and allows for the design of higher-order G4 DNA structures. To understand the molecular driving forces for G4 association, here, we study the binding interaction between two parallel-stranded G-quadruplexes using all-atom molecular dynamics simulations. The predicted dimerization free energies show that direct binding through the 5'-G-tetrads is the most preferred of all possible end-to-end stacking orientations, consistently with all available experimental data. Decomposition of dimerization enthalpies in combination with simulations at varying ionic strength further indicate that the observed orientational preferences arise from a fine balance between the electrostatic repulsion of the sugar-phosphate backbones and favorable counterion binding at the dimeric interface. We also demonstrate how these molecular-scale findings can be used to devise means of controlling G4 dimerization equilibrium, e.g., by altering salt concentration and using G4-targeted ligands.

Parallel G-triplexes and G-hairpins as potential transitory ensembles in the folding of parallel-stranded DNA G-Quadruplexes.

Guanine quadruplexes (G4s) are non-canonical nucleic acids structures common in important genomic regions. Parallel-stranded G4 folds are the most abundant, but their folding mechanism is not fully understood. Recent research highlighted that G4 DNA molecules fold via kinetic partitioning mechanism dominated by competition amongst diverse long-living G4 folds. The role of other intermediate species such as parallel G-triplexes and G-hairpins in the folding process has been a matter of debate. Here, we use standard and enhanced-sampling molecular dynamics simulations (total length of ∼0.9 ms) to study these potential folding intermediates. We suggest that parallel G-triplex per se is rather an unstable species that is in local equilibrium with a broad ensemble of triplex-like structures. The equilibrium is shifted to well-structured G-triplex by stacked aromatic ligand and to a lesser extent by flanking duplexes or nucleotides. Next, we study propeller loop formation in GGGAGGGAGGG, GGGAGGG and GGGTTAGGG sequences. We identify multiple folding pathways from different unfolded and misfolded structures leading towards an ensemble of intermediates called cross-like structures (cross-hairpins), thus providing atomistic level of description of the single-molecule folding events. In summary, the parallel G-triplex is a possible, but not mandatory short-living (transitory) intermediate in the folding of parallel-stranded G4.

NMR solution and X-ray crystal structures of a DNA molecule containing both right- and left-handed parallel-stranded G-quadruplexes.

Analogous to the B- and Z-DNA structures in double-helix DNA, there exist both right- and left-handed quadruple-helix (G-quadruplex) DNA. Numerous conformations of right-handed and a few left-handed G-quadruplexes were previously observed, yet they were always identified separately. Here, we present the NMR solution and X-ray crystal structures of a right- and left-handed hybrid G-quadruplex. The structure reveals a stacking interaction between two G-quadruplex blocks with different helical orientations and displays features of both right- and left-handed G-quadruplexes. An analysis of loop mutations suggests that single-nucleotide loops are preferred or even required for the left-handed G-quadruplex formation. The discovery of a right- and left-handed hybrid G-quadruplex further expands the polymorphism of G-quadruplexes and is potentially useful in designing a left-to-right junction in G-quadruplex engineering.

Preferential Binding of π-Ligand Porphyrin Targeting 5'-5' Stacking Interface of Human Telomeric RNA G-Quadruplex Dimer.

Human telomeric RNA (TERRA) containing thousands of G-rich repeats has the propensity to form parallel-stranded G-quadruplexes. The emerging crucial roles of TERRA G-quadruplexes in RNA biology fuel increasing attention for studying anticancer ligand binding with such structures, which, however, remains scarce. Here we utilized multiple steady-state and time-resolved spectroscopy analyses in conjunction with NMR methods and investigated thoroughly the binding behavior of TMPyP4 to a TERRA G-quadruplex dimer formed by the 10-nucleotide sequence r(GGGUUAGGGU). It is clearly identified that TMPyP4 intercalates into the 5'-5' stacking interface of two G-quadruplex blocks with a binding stoichiometry of 1:1 and binding constant of 1.92 × 106 M-1. This is consistent with the unique TERRA structural features of the enlarged π-π stacking plane of the A·(G·G·G·G)·A hexad at 5'-ends of each G-quadruplex block. The preferential binding of π-ligand porphyrin to the 5'-5' stacking interface of the native TERRA G-quadruplex dimer is first ascertained by the combination of dynamics and structural characterization.

Fast detection of mycoplasma pneumoniae by interaction of tetramolecular G-quadruplex with graphene oxide

Fast and sensitive detection of mycoplasma pneumoniae (MP) is quite momentous in early diagnosis of respiratory infections. Herein, we present a new biosensing platform for MP based on the interaction of a designed parallel-stranded tetrapod G-quadruplex (TP-G4) with graphene oxide. The G-quadruplex is formed with four fluorescent dye labeled ssDNA molecules, which is demonstrated with gel electrophoresis, CD spectra and fluorescence spectra. The interaction induces the adsorption of TP-G4 on graphene oxide, which leads to the fluorescence quenching of the dye. Upon the recognition of the adsorbed ends to target MP, the TP-G4 can be released from graphene oxide surface, producing a “signal-on” fluorescence biosensor for MP. The designed biosensing strategy shows a detection limit of 3.96 nM and an analytical time of 10 min. The facile and fast operation process and acceptable sensitivity meet the demands of early point-of-care testing of mycoplasma pneumoniae, showing its promising application in clinic diagnosis.

Case studies on potential G-quadruplex-forming sequences from the bacterial orders Deinococcales and Thermales derived from a survey of published genomes.

Genomes provide a platform for storage of chemical information that must be stable under the context in which an organism thrives. The 2‘-deoxyguanosine (G) nucleotide has the potential to provide additional chemical information beyond its Watson-Crick base-pairing capacity. Sequences with four or more runs of three G nucleotides each are potential G-quadruplex forming sequences (PQSs) that can adopt G-quadruplex folds. Herein, we analyzed sequenced genomes from the NCBI database to determine the PQS densities of the genome sequences. First, we found organisms with large genomes, including humans, alligators, and maize, have similar densities of PQSs (~300 PQSs/Mbp), and the genomes are significantly enriched in PQSs with more than four G tracks. Analysis of microorganism genomes found a greater diversity of PQS densities. In general, PQS densities positively tracked with the GC% of the genome. Exceptions to this observation were the genomes from thermophiles that had many more PQSs than expected by random chance. Analysis of the location of these PQSs in annotated genomes from the order Thermales showed these G-rich sequences to be randomly distributed; in contrast, in the order Deinococcales the PQSs were enriched and biased around transcription start sites of genes. Four representative PQSs, two each from the Thermales and Deinococcales, were studied by biophysical methods to establish the ability of them to fold to G-quadruplexes. The experiments found the two PQSs in the Thermales did not adopt G-quadruplex folds, while the two most common in the Deinococcales adopted stable parallel-stranded G-quadruplexes. The findings lead to a hypothesis that thermophilic organisms are enriched with PQSs as an unavoidable consequence to stabilize thermally their genomes to live at high temperature; in contrast, the genomes from stress-resistant bacteria found in the Deinococcales may utilize PQSs for gene regulatory purposes.