Telomeric G-quadruplex Research Articles

Characterization of human telomere RNA G-quadruplex structures in vitro and in living cells using 19F NMR spectroscopy.

Human telomeric RNA has been identified as a key component of the telomere machinery. Recently, the growing evidence suggests that the telomeric RNA forms G-quadruplex structures to play an important role in telomere protection and regulation. In the present studies, we developed a 19F NMR spectroscopy method to investigate the telomeric RNA G-quadruplex structures in vitro and in living cells. We demonstrated that the simplicity and sensitivity of 19F NMR approach can be used to directly observe the dimeric and two-subunits stacked G-quadruplexes in vitro and in living cells and quantitatively characterize the thermodynamic properties of the G-quadruplexes. By employing the 19F NMR in living cell experiment, we confirmed for the first time that the higher-order G-quadruplex exists in cells. We further demonstrated that telomere RNA G-quadruplexes are converted to the higher-order G-quadruplex under molecular crowding condition, a cell-like environment. We also show that the higher-order G-quadruplex has high thermal stability in crowded solutions. The finding provides new insight into the structural behavior of telomere RNA G-quadruplex in living cells. These results open new avenues for the investigation of G-quadruplex structures in vitro and in living cells.

Enhanced G-Quadruplex DNA Stabilization and Telomerase Inhibition by Novel Fluorescein Derived Salen and Salphen Based Ni(II) and Pd(II) Complexes.

Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work, we have synthesized nickel(II) and palladium(II) salen and salphen complexes by using readily available fluorescein as the backbone to provide an extended aromatic surface. The metal complexes exhibit affinity toward the human telomeric G-quadruplex DNA with promising inhibition of telomerase activity. This has been ascertained by their efficiency in the long term cell proliferation assay which showed significant cancer cell toxicity in the presence of the metal complexes. Confocal microscopy showed cellular internalization followed by localization in the nucleus and mitochondria. Considerable population at the sub-G1 phase of the cell cycle showed cell death via apoptotic pathway.

MD Simulations and CD Spectroscopies of (Benz)Acridine: rDNA G-Quadruplex Complexes

In both prokaryotic and eukaryotic cells, guanine-rich stretches of DNA sequences are found in gene regulatory regions and telomeric DNAs to form G-quadruplexes, and they are involved in many fundamental biological processes and have been implicated in a number of diseases including cancer. While most previous G-quadruplex studies have focused on telomeric G-quadruplexes for validating targets for anticancer therapy, the nucleolar G-quadruplexes may be an even more promising target for chemotherapeutic intervention. The nucleolus plays a central role in tumor pathology because it controls the high level of ribosomal RNA (rRNA) synthesis necessary to sustain ribosome biogenesis during rapid cancer cell growth. Ribosome biogenesis occurs in the nucleolus and is tightly regulated by many cell signaling pathways that converge on the RNA polymerase I complex (Pol I). Using small molecules (drugs) to disrupt ribosome biogenesis represents an attractive strategy for selectively killing cancer cells. Aromatic acridines that sit on top of a G-quadruplex tetrad via π-π stacking interactions have been extensively explored as scaffolds for developing G-quadruplex-specific binders. In this present study, we performed MD simulations of 13 modeled putative rDNA G-quadruplexes that have been characterized via CD spectra to form G-quadruplexes. In addition, we simulated their interactions with designed acridines and benzacridines and analyzed them using a set of novel order parameters to quantify their binding selectivities. We are currently validating our computational predictions via CD spectra. In one G-quadruplex we studied, NUC 23, CD spectra shows that it adopts a parallel topology structure that is further stabilized upon interactions with benzacridine. In excellent agreement, we also observe the same phenomenon in our MD simulations.

Naphthalene diimide-polyamine hybrids as antiproliferative agents: Focus on the architecture of the polyamine chains

Naphthalene diimides (NDIs) have been widely used as scaffold to design DNA-directed agents able to target peculiar DNA secondary arrangements endowed with relevant biochemical roles. Recently, we have reported disubstituted linear- and macrocyclic-NDIs that bind telomeric and non-telomeric G-quadruplex with high degree of affinity and selectivity. Herein, the synthesis, biological evaluation and molecular modelling studies of a series of asymmetrically substituted NDIs are reported. Among these, compound 9 emerges as the most interesting of the series being able to bind telomeric G-quadruplex (ΔTm = 29 °C at 2.5 μM), to inhibit the activity of DNA processing enzymes, such as topoisomerase II and TAQ-polymerase, and to exert antiproliferative effects in the NCI panel of cancer cell lines with GI50 values in the micro-to nanomolar concentration range (i.e. SR cell line, GI50 = 76 nM). Molecular mechanisms of cell death have been investigated and molecular modelling studies have been performed in order to shed light on the antiproliferative and DNA-recognition processes.

Effects of the central potassium ions on the G-quadruplex and stabilizer binding

Human telomeres undertake the structure of intra-molecular parallel G-quadruplex in the presence of K+ in eukaryotic cell. Stabilization of the telomere G-quadruplex represents a potential strategy to prevent telomere lengthening by telomerase in cancer therapy. Current work demonstrates that the binding of central K+ with the parallel G-quadruplex is a coordinated water directed step-wise process. The K+ above the top G-tetrad is prone to leak into environment and the 5′-adenine quickly flips over the top G-tetrad, leading to the bottom gate of G-tetrads as the only viable pathway of K+ binding. Present molecular dynamics studies on the two most potent stabilizers RHPS4 and BRACO-19 reveal that the central K+ has little influence on the binding conformations of the bound stabilizers. But without the central K+, either RHPS4 or BRACO-19 cannot stabilize the structure of G-quadruplex. The binding strength of stabilizers evaluated by the MM-PBSA method follows the order of BRACO-19> RHPS4, which agrees with the experimental results. The difference in binding affinities between RHPS4 and BRACO-19 is probably related to the ability to form intramolecular hydrogen bonds and favorable van del Waals interactions with G-quadruplex. In the models that have one central K+ located at the upper/lower binding site, the corresponding top/bottom stacked stabilizers show more favorable binding affinities, indicating the apparent promoting effect of central K+ on the stabilizer binding. Our findings provide further insights into the regulatory effect of K+ on the G-quadruplex targeted binding, which is meaningful to the development of G-quadruplex stabilizers.

Assessment of human telomeric G-quadruplex structures using surface-enhanced Raman spectroscopy.

G-Quadruplex (G4) structures of a human telomeric 24-mer (5'-TTAGGGTTAGGGTTAGGGTTAGGG-3') sequence (Tel24) stabilized by sodium and potassium ions have been assessed using surface-enhanced Raman scattering (SERS) spectroscopy. The distinctive SERS spectra of Tel24 in the presence of 100mM Na+ and 100mM K+ were obtained and the SERS bands characteristic of the antiparallel basket-type and the mixed hybrid (3+1) structures, respectively, were identified and assigned. The influence of the SERS - active substrate on the scattering enhancement was studied using citrate- and chloride-covered silver nanoparticles, in the absence and presence of the aggregating agent (0.1M Na2SO4 and 0.1M K2SO4). The highly reproducible SERS spectra of Tel24 obtained in various SERS active media indicated the same adsorption mechanism of the cation - stabilized G-quadruplexes onto the metal surface, regardless of the silver colloid. The remarkable resemblance between the circular dichroism (CD) spectra of the Tel24 structures with and without the colloid confirmed that interaction with the enhancing silver surface did not affectthe stability of the formed G4 structures. The presented study pointed to a great potential of the SERS spectroscopy for the sensitive structural analysis of various G4 topologies. Graphical Abstract SERS spectroscopy allowed identification of Na+ stabilized antiparallel basket-type and K+ stabilized hybrid (3+1) structures of the same 24-mer human telomeric sequence.

Tetrazolylpyrene unnatural nucleoside as a human telomeric multimeric G-quadruplex selective switch-on fluorescent sensor.

We report herein the specific sensing of dimeric H45 G-quadruplex DNA via a fluorescence light-up response using fluorescent tetrazolylpyrene nucleoside (TzPyBDo) as a probe. The strong binding of the probe via an intercalative stacking interaction inside the connecting loop of two G-quadruplex units of H45 and the discrimination to other monomeric and long DNA duplexes are accompanied by a drastic enhancement of the emission intensity without compromising the conformation and stability.

Human Telomeric G-Quadruplex Structures and G-Quadruplex-Interactive Compounds.

G-quadruplexes are noncanonical secondary structures formed in DNA sequences containing consecutive runs of guanines. It has been shown that the 3' G-rich single-stranded overhangs of human telomeres can form G-quadruplex structures, and the human telomeric DNA G-quadruplexes are considered attractive targets for anticancer drugs. G-quadruplex-interactive compounds have been shown to inhibit telomerase access as well as telomere capping. Nuclear magnetic resonance (NMR) spectroscopy is a powerful method in determining the G-quadruplex structures under physiologically relevant conditions. We present the NMR and biophysical methodology used in our research group for the study of G-quadruplex structures in physiologically relevant solution and their interactions with small-molecule compounds.

Sedimentation velocity analysis of TMPyP4-induced dimer formation of human telomeric G-quadruplex

Analytical ultracentrifugation sedimentation velocity (AUC-SV) was used to study the interactions between TMPyP4 and AGGG(TTAGGG)3 (Tel22) and the TMPyP4-induced dimer formation of G-quadruplex.

Design, synthesis and antimalarial activity of novel bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives

Novel series of bis- and tris-pyrrolo[1,2-a]quinoxaline derivatives 1 were synthesized and tested for in vitro activity upon the intraerythrocytic stage of W2 and 3D7 Plasmodium falciparum strains. Biological results showed good antimalarial activity with IC50 in the μM range. In attempting to investigate the large broad-spectrum antiprotozoal activities of these new derivatives, their properties toward Leishmania donovani were also investigated and revealed their selective antiplasmodial profile. In parallel, the in vitro cytotoxicity of these molecules was assessed on the human HepG2 cell line. Structure–activity relationships of these new synthetic compounds are discussed here. The bis-pyrrolo[1,2-a]quinoxalines 1n and 1p were identified as the most potent antimalarial candidates with selectivity index (SI) of 40.6 on W2 strain, and 39.25 on 3D7 strain, respectively. As the telomeres of the parasite could constitute an attractive target, we investigated the possibility of targeting Plasmodium telomeres by stabilizing the Plasmodium telomeric G-quadruplexes through a FRET melting assay by our new compounds.

Binding modes and pathway of RHPS4 to human telomeric G-quadruplex and duplex DNA probed by all-atom molecular dynamics simulations with explicit solvent.

RHPS4, a potent binder to human telomeric DNA G-quadruplex, shows high efficacy in tumor cell growth inhibition. However, it's preferential binding to DNA G-quadruplex over DNA duplex (about 10 fold) remains to be improved toward its clinical application. A high resolution structure of the single-stranded telomeric DNA G-quadruplexes, or B-DNA duplex, in complex with RHPS4 is not available yet, and the binding nature of this ligand to these DNA forms remains to be elusive. In this study, we carried out 40 μs molecular dynamics binding simulations with a free ligand to decipher the binding pathway of RHPS4 to a DNA duplex and three G-quadruplex folders (parallel, antiparallel and hybrid) of the human telomeric DNA sequence. The most stable binding mode identified for the duplex, parallel, antiparallel and hybrid G-quadruplexes is an intercalation, bottom stacking, top intercalation and bottom intercalation mode, respectively. The intercalation mode with similar binding strength to both the duplex and the G-quadruplexes, explains the lack of binding selectivity of RHPS4 to the G-quadruplex form. Therefore, a ligand modification that destabilizes the duplex intercalation mode but stabilizes the G-quadruplex intercalation mode will improve the binding selectivity toward G-quadruplex. The intercalation mode of RHPS4 to both the duplex and the antiparallel and the hybrid G-quadruplex follows a base flipping-insertion mechanism rather than an open-insertion mechanism. The groove binding, the side binding and the intercalation with flipping out of base were observed to be intermediate states before the full intercalation state with paired bases.

Restriction of telomerase capping by short non-toxic peptides via arresting telomeric G-quadruplex

The stabilization of a G-quadruplex structure in human telomeric DNA has become a promising strategy in the development of cancer therapeutics.

NMR based model of human telomeric repeat G-quadruplex in complex with 2,4,6-triarylpyridine family ligand.

G-quadruplexes (G4) are one of the several different forms of non-canonical DNA structures that can occur in our genome. Their existence is thought to be implicated in important biological functions such as positive and negative transcription regulation or telomeric extension. The human telomeric sequence G4 formed by repetitive nucleotide sequences (T2AG3) at each chromosome end is an important example of intramolecular G4. Knowing the atomic details for different families of ligands targeting G-quadruplex structures hypothetically found in the telomeric repeat it is an important step for rational drug design. Especially if the aim is to prevent or interfere with telomerase extending the 3' end of telomeres. In this study, we report the structure of the complex formed between the telomeric repeat sequence (d[AG3(T2AG3)3]) intramolecular G-quadruplex and the 2,4,6-Triarylpyridine compound. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Specific targeting of telomeric multimeric G-quadruplexes by a new triaryl-substituted imidazole.

Multiple G-quadruplex units in the 3΄-terminal overhang of human telomeric DNA can associate and form multimeric structures. The specific targeting of such distinctive higher-order G-quadruplexes might be a promising strategy for developing selective anticancer agents with fewer side effects. However, thus far, only a few molecules were found to selectively bind to telomeric multimeric G-quadruplexes, and their effects on cancer cells were unknown. In this study, a new triaryl-substituted imidazole derivative called IZNP-1 was synthesized and found to specifically bind to and strongly stabilize telomeric multimeric G-quadruplexes through intercalating into the pocket between the two quadruplex units. The pocket size might affect the binding behavior of IZNP-1. Further cellular studies indicated that IZNP-1 could provoke cell cycle arrest, apoptosis and senescence in Siha cancer cells, mainly because of telomeric DNA damage and telomere dysfunction induced by the interactions of IZNP-1 with telomeric G-quadruplexes. Notably, IZNP-1 had no effect on the transcriptional levels of several common oncogenes that have the potential to form monomeric G-quadruplex structures in their promoter regions. Such behavior differed from that of traditional telomeric G-quadruplex ligands. Accordingly, this work provides new insights for the development of selective anticancer drugs targeting telomeric multimeric G-quadruplexes.

Cyclic ferrocenylnaphthalene diimide derivative as a new class of G-quadruplex DNA binding ligand

To identify an effective ligand that binds to a G-quadruplex structure but not a double-stranded DNA (dsDNA), a set of biophysical and biochemical experiments were carried out using newly synthesized cyclic ferrocenylnaphthalene diimide (cFNDI, 1) or the non-cyclic derivative (2) with various structures of G-quadruplex DNAs and dsDNA. Compound 1 bound strongly to G-quadruplexes DNAs (106M−1 order) with diminished binding to dsDNA (104M−1 order) in 100mM AcOH-AcOK buffer (pH 5.5) containing 100mM KCl. Interestingly, 1 showed an approximately 50-fold higher selectivity to mixed hybrid-type telomeric G-quadruplex DNA (K=3.4×106M−1 and a 2:1 stoichiometry) than dsDNA (K=7.5×104M−1) did. Furthermore, 1 showed higher thermal stability to G-quadruplex DNAs than it did to dsDNA with a preference for c-kit and c-myc G-quadruplex DNAs over telomeric and thrombin binding aptamers. Additionally, 1 exhibited telomerase inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.4μM. Compound 2 showed a preference for G-quadruplex; however, the binding affinity magnitude and preference were improved in 1 because the former had a cyclic structure.

Oxoisoaporphine as Potent Telomerase Inhibitor.

Two compounds previously isolated from traditional Chinese medicine, Menispermum dauricum (DC), 6-hydroxyl-oxoisoaporphine (H-La), and 4,6-di(2-pyridinyl)benzo[h]isoindolo[4,5,6-de]quinolin-8(5H)-one (H-Lb), were known to have in vitro antitumor activity and to selectively bind human telomeric, c-myc, and bcl-2 G-quadruplexes (G4s). In this study, the binding properties of these two compounds to telomerase were investigated through molecular docking and telomeric repeat amplication protocol and silver staining assay (TRAP-silver staining assay). The binding energies bound to human telomerase RNA were calculated by molecular docking to be −6.43 and −9.76 kcal/mol for H-La and H-Lb, respectively. Compared with H-La, the ligand H-Lb more strongly inhibited telomerase activity in the SK-OV-3 cells model.

Downregulation of oncogenic RAS and c-Myc expression in MOLT-4 leukaemia cells by a salicylaldehyde semicarbazone copper(II) complex

Copper complexes with potent anti-tumor effect have been extensively developed. Most investigations of their modes of action focused on the biomolecular targets but not the signal transduction between target binding and cell death. We have previously shown that the cytotoxic complex pyridine(2,4-dihydroxybenzaldehyde dibenzyl semicarbazone)copper(II) (complex 1) shows selective binding to human telomeric G-quadruplex DNA over double-stranded DNA in vitro. Herein, we elucidate the mechanism of action by which complex 1 induces apoptosis in MOLT-4 cells. Complex 1 accumulates in the nuclei and differentially downregulates the expression of c-Myc, c-Kit and KRAS oncogenes. Chemical affinity capture assay results show that the complex is associated with c-Myc and KRAS quadruplex sequences in MOLT-4 cells. We further showed that the reduction in Ras protein expression resulted in attenuated MEK-ERK and PI3K-Akt signalling activities, leading to the activation of caspase-dependent apoptosis. Notably, complex 1 increased the sensitivity of MOLT-4 cells to cisplatin and vice versa. Overall, we demonstrated that complex 1 induces apoptosis, at least in part, by suppressing KRAS, c-Kit and c-Myc oncogene expression and the pro-survival MEK-ERK and PI3K-Akt signalling pathways.

Coarse-Grained Simulations Complemented by Atomistic Molecular Dynamics Provide New Insights into Folding and Unfolding of Human Telomeric G-Quadruplexes.

G-quadruplexes are the most important noncanonical DNA architectures. Many quadruplex-forming sequences, including the human telomeric sequence d(GGGTTA)n, have been investigated due to their implications in cancer and other diseases, and because of their potential in DNA-based nanotechnology. Despite the availability of atomistic structural studies of folded G-quadruplexes, their folding pathways remain mysterious, and mutually contradictory models of folding coexist in the literature. Recent experiments convincingly demonstrated that G-quadruplex folding often takes days to reach thermodynamic equilibrium. Based on atomistic simulations of diverse classes of intermediates in G-quadruplex folding, we have suggested that the folding is an extremely multipathway process combining a kinetic partitioning mechanism with conformational diffusion. However, complete G-quadruplex folding is far beyond the time scale of atomistic simulations. Here we use high-resolution coarse-grained simulations to investigate potential unfolding intermediates, whose structural dynamics are then further explored with all-atom simulations. This multiscale approach indicates how various pathways are interconnected in a complex network. Spontaneous conversions between different folds are observed. We demonstrate the inability of simple order parameters, such as radius of gyration or the number of native H-bonds, to describe the folding landscape of the G-quadruplexes. Our study also provides information relevant to further development of the coarse-grained force field.

Targeting human telomeric and c-myc G-quadruplexes with alkynylplatinum(II) terpyridine complexes under molecular crowding conditions

The interactions between alkynylplatinum(II) terpyridine complexes 1–3 and the G-quadruplex DNA, including c-myc and telomeric quadruplex DNA, are investigated both in dilute solution and under molecular crowding conditions. The UV–vis absorption spectroscopy, circular dichroism and molecular docking studies suggest that 1–3 associate with telomeric and c-myc G-quadruplexes via groove binding, and electrostatic interactions. Experimental studies indicate that under molecular crowding conditions (in the presence of 40wt% PEG 200), 1–2 show weak affinity for c-myc, while 3 still displays high affinity and selectivity for c-myc. On the other hand, 1–3 act as efficient and selective ligand for telomeric quadruplex DNA under molecular crowding conditions. The complex 3 exhibits excellent cytotoxicity against A549, K562 and SGC-7901, with IC50 values that are 35.0-fold, 10.0-fold, and 12.1-fold lower than the values of cisplatin under the same conditions, respectively.

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes.

G-quadruplexes (G4s) are DNA secondary structures that are capable of forming and function in vivo. The propensity of G4s to exhibit extreme polymorphism and complex dynamics is likely to influence their cellular function, yet a clear microscopic picture of their folding process is lacking. Here we employed single-molecule FRET microscopy to obtain a direct view of the folding and underlying conformational dynamics of G4s formed by the human telomeric sequence in potassium containing solutions. Our experiments allowed detecting several folded states that are populated in the course of G4 folding and determining their folding energetics and timescales. Combining the single-molecule data with molecular dynamics simulations enabled obtaining a structural description of the experimentally observed folded states. Our work thus provides a comprehensive thermodynamic and kinetic description of the folding of G4s that proceeds through a complex multi-route pathway, involving several marginally stable conformational states.