Human Telomeric G-quadruplex Research Articles

Synergically Remodeling Human Telomeric G-Quadruplexes into DNA Mimics of GFP with a Na+ Selectivity

Synergically Remodeling Human Telomeric G-Quadruplexes into DNA Mimics of GFP with a Na⁺ Selectivity

Rationally Designed G-Quadruplex Selective "Turn-On" NIR Fluorescent Probe with Large Stokes Shift for Nucleic Acid Research-Based Applications.

Guanine-rich DNA/RNA sequences can form Hoogsteen bonds to adopt noncanonical secondary structures called G-quadruplexes, and these have been associated with diverse cellular processes. There has been considerable research interest in the design of G4-interacting ligands for cellular probing of the G4 structure and understanding its associated biological function. Most of the fluorescent G4 ligands either do not have significant selectivity over other nucleic acid structures, have high Stokes shift, or are not in the near-infrared (NIR) region, which limits its cellular visualization. The current work involves the rational design and synthesis of NIR fluorescent probes comprising a (Z)-1-methyl-2-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium scaffold. Among the designed molecules, 4a exhibited far-red fluorescence (λmax = 680 nm) with large Stokes shift (∼182 nm) upon selective binding to human telomeric G-quadruplexes. The dye 4a does not disturb the conformation and stability of G-quadruplexes, thereby making it suitable for nucleic acid research based applications. Interestingly, 4a showed remarkable selectivity over single- and double-stranded structures in contrast to a commercially available quadruplex binding probe, Thiazole orange (TO). The molecular docking studies indicate that 4a binds at the groove region of the telomeric DNA G-quadruplex through π-π stacking interactions with the quinoline and amine-substituted phenyl ring and with the phosphate backbone through anion-π interactions with the benzothiazole ring. The designed molecule 4a has interesting photophysical properties, cell permeability, and biocompatibility with minimal cytotoxicity. Fluorescence imaging studies in live HeLa cells showed that probe 4a binds to the transient population of the DNA G-quadruplex in the nucleus and RNA quadruplexes in the cytoplasm. In brief, G-quadruplex NIR fluorescent probe 4a with a higher signal/noise ratio has significant potential for cellular imaging studies and thus opens avenues to decipher the biological pathways for better understanding of G-quadruplex biology.

Enhanced Recognition of a Herbal Compound Epiberberine by a DNA Quadruplex-Duplex Structure.

The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is an excellent EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.

Structural and biophysical insights into the interactions of anisotropic gold nanoparticles with human telomeric G-quadruplex DNA: Spectroscopic and calorimetric approach

We are reporting curcumin-induced synthesis of anisotropic citrate capped Gold nanoparticles (ctGNPs). The techniques such as UV–visible spectroscopy, Raman spectroscopy, FT-IR, X-ray diffraction (XRD), and Transmission Electron Microscopy (TEM) were used to characterize the nanoparticles. The synthetic route shows the formation of an anisotropic gold nanostructure consisting of spherical, triangles, hexagonals, and a low-yield rod with an aspect ratio ranging from 4.2 to 8.5. Curcumin-derived ctGNPs shows stability at different physiological conditions and better biocompatibility. Synthesized nanoparticles are found non-toxic towards eukaryotic cells but more effective against the cancer cell lines HeLa and MCF-7. The interaction of these synthesized nanoparticles with human telomeric G-quadruplex (GQ) DNA was studied using different physiochemical methods. The spectroscopic studies show that synthesized nanoparticles have stronger binding affinity towards telomeric GQ as compared to ds DNA through Van der Waals and H-bonding interactions. Thermodynamic interpretation reveals that the formation of the complex between the telomeric GQ and ctGNPs are enthalpy driven and entropy unfavourable process, resulting in motion freezing and, eventually, AuNP aggregation. Thus, our study shows a new approach to understand the interaction of telomeric G-quadruplexes with gold nanoparticles generated via the green route.

Flanking Effect on the Structure and Stability of Human Telomeric G-Quadruplex in Varying Salt Concentrations.

The stability of the human telomere G-quadruplex (G4) is directly linked to cancer disease. The human telomere is mostly associated with the flanking nucleobases, which can affect the stability of G4. Hence, in this study, the effect of the flanking nucleobases in the context of their chemical nature, number, and position on the structure and stability of G4 has been investigated in varying concentrations of KCl mimicking the normal and cancer KCl microenvironments. The addition of flanking nucleobases does not alter the G4 topology. However, the presence of merely a single flanking nucleobase destabilizes the telomeric G4. This destabilizing effect is more prominent for thymine than adenine flanking nucleobase, probably due to the formation of the intermolecular G4 topology by thymine. Interestingly, the change in the stability of the telomeric G4 in the presence of thymine flanking nucleobase is sensitive to the concentration of KCl relevant to the normal and cancerous microenvironments, in contrast to adenine. Flanking nucleobases have a greater impact at the 5' end compared to the 3' end, particularly noticeable in KCl concentrations resembling the normal microenvironment rather than the cancerous one. These findings indicate that the effect of the flanking nucleobases on telomeric G4 is different in the KCl salt relevant to normal and cancerous microenvironments. This study may be helpful in attaining molecular-level insight into the role of G4 in telomeric length regulation under normal and cancerous KCl salt conditions.

Dynamic Structures and Fast Transition Kinetics of Oxidized G-Quadruplexes.

8-oxoguanines (8-oxoG) in cells form compromised G-quadruplexes (GQs), which may vary GQ mediated gene regulations. By mimicking molecularly crowded cellular environment using 40% DMSO or sucrose, here it is found that oxidized human telomeric GQs have stabilities close to the wild-type (WT) GQs. Surprisingly, while WT GQs show negative formation cooperativity between a Pt(II) binder and molecularly crowded environment, positive cooperativity is observed for oxidized GQ formation. Single-molecule mechanical unfolding reveals that 8-oxoG sequence formed more diverse and flexible structures with faster folding/unfolding transition kinetics, which facilitates the Pt(II) ligand to bind the best-fit structures with positive cooperativity. These findings offer new understanding on structures and properties of oxidized G-rich species in crowded environments. They also provide insights into the design of better ligands to target oxidized G-rich structures formed under oxidative cell stress.

Electrochemical genosensor based on RNA-responsive human telomeric G-quadruplex DNA: A proof-of-concept with SARS-CoV-2 RNA

In this report, a facile and label-free electrochemical RNA biosensor is developed by exploiting methylene blue (MB) as an electroactive positive ligand of G-quadruplex. The electrochemical response mechanism of the nucleic acid assay was based on the change in differential pulse voltammetry (DPV) signal of adsorbed MB on the immobilized human telomeric G-quadruplex DNA with a loop that is complementary to the target RNA. Hybridization between synthetic positive control RNA and G-quadruplex DNA probe on the transducer platform rendered a conformational change of G-quadruplex to double-stranded DNA (dsDNA), and increased the redox current of cationic MB π planar ligand at the sensing interface, thereby the electrochemical signal of the MB-adsorbed duplex is proportional to the concentration of target RNA, with SARS-CoV-2 (COVID-19) RNA as the model. Under optimal conditions, the target RNA can be detected in a linear range from 1 zM to 1 μM with a limit of detection (LOD) obtained at 0.59 zM for synthetic target RNA and as low as 1.4 copy number for positive control plasmid. This genosensor exhibited high selectivity towards SARS-CoV-2 RNA over other RNA nucleotides, such as SARS-CoV and MERS-CoV. The electrochemical RNA biosensor showed DPV signal, which was proportional to the 2019-nCoV_N_positive control plasmid from 2 to 200000 copies (R2 = 0.978). A good correlation between the genosensor and qRT-PCR gold standard was attained for the detection of SARS-CoV-2 RNA in terms of viral copy number in clinical samples from upper respiratory specimens.

Probing the binding and interaction of curcumin-tryptophan conjugate to G-quadruplex motif via spectroscopic and molecular docking approach

A potential ligand with the ability to interact with the structure of G-quadruplexes has been promising strategy for develop anticancer drug. Curcumin, a natural antioxidant is known to induce apoptosis in the tumour cells and was previously found as G-quadruplex binder. We investigated how the conjugation of tryptophan with curcumin affects its ability as G-quadruplex DNA binder. We have reported the differential interaction of Curcumin-tryptophan (CT) with a parallel G-quadruplex(GQ) formed by the nuclease hypersensitivity element (NHEIII) Pu27 present of the protooncogene and human telomeric G-quadruplex sequence. Taken into account, ligand CT has a stronger binding affinity for the G-quadruplex over the canonical duplex DNA. We have used biophysical approach such as uv-visible, fluorescence, circular dichroism, differential scanning calorimetry and molecular docking experiment to show that curcumin tryptophan interacts with the G-quadruplex. The results indicate that CT has a binding affinity of 106 M−1 for G-quadruplexes. Thermodynamic studies show that the ligand CT destabilizes the G-quadruplexes (6–8 °C) due to aromatic core stacking between the tetrad. The anti-proliferative experiment of the synthesized ligand CT in vitro on several cancer cell lines such as HeLa cells and MCF-7 cell lines shows that it is more effective against MCF-7. Thus, present studies report Curcumin-tryptophan as a potential G-quadruplex binder that destabilizes the quadruplex structure .

NRAS DNA G-quadruplex-targeting molecules for sequence-selective enzyme inhibition.

Sequence-selective G-quadruplex ligands are valuable for controlling gene expression. Here, we established a new fluorescence displacement assay using a NRAS G-quadruplex selective fluorescent probe to identify sequence-selective DNA G-quadruplex ligands. These sequence-selective NRAS G-quadruplex ligands retained their binding affinity even in the presence of excessive human telomeric DNA G-quadruplex and regulated enzymatic activities in a sequence-selective manner.

Spectroscopy and calorimetry evidence of binding-induced thermal stabilization of human telomeric G-quadruplex DNA by 1,5-disubstituted morpholino-amido anthraquinone: Molecular basis of anticancer action

We report interaction of 1,5-Bis (3-morpholinopropionamido) anthracene-9,10-dione with Human Telomeric G4 DNA sequences, wHTel26 and HTel22 in presence of K+ and Na+ ions, respectively. Surface Plasmon Resonance yields association constant Kb = 1.0–3.8x105 M−1 with HTel22 in K+-rich solution. Significant hypochromism (46–79 %) and fluorescence quenching (83–92 %), no change in absorption and emission maxima, unaltered fluorescence lifetime of ligand, change in conformation of G4 DNA with no overall disruption of G-quartets and absence of induced Circular Dichroism band, suggest external groove binding of ligand. Molecular docking shows short contacts of 9O, 15N, 18O, 11NH, 12CO atoms of ligand with A3, G4, G22, G23 and A1, G3, G14 bases of 3 + 1 hybrid-2 and 2 + 2 anti-parallel conformers, respectively without making any contact with loops, to an energetically more favourable conformation. Ligand-influenced thermal stabilization, up to ΔTm = 37/48 °C, may disrupt access of telomerase enzyme to telomere and promote apoptosis in malignant tissues.

Recognition of human telomeric G-quadruplex DNA by 1,5-disubstituted diethyl-amido anthraquinone derivative in different ion environments causing thermal stabilization and apoptosis

Ligand binding to G-quadruplex (G4) structures at human telomeric DNA ends promotes thermal stabilization, disrupting the interaction of the telomerase enzyme, which is found active in 80–85% of cancers and serves as a molecular marker. Anthraquinone compounds are well-known G-quadruplex (G4) binders that inhibit telomerase and induce apoptosis in cancer cells. Our current investigation is based on 1,5-bis[3-(diethylamino)propionamido]anthracene-9,10-dione, a derivative of anthraquinone and its binding characterization with two different human telomeric DNA structures, wHTel26 and HTel22, in the effect of K+ and Na+ by using an array of biophysical, calorimetry, molecular docking and cell viability assay techniques. Binding constants (K b) in the range of ∼105–107 M−1 and stoichiometries of 1:1, 2:1 & 4:1 were obtained from the absorbance, fluorescence, and circular dichroism study. Remarkable hypochromism (55, 97%) and ∼17 nm shift in absorbance, fluorescence quenching (95, 97%), the unaltered value of fluorescence lifetime, restoration of Circular Dichroism bands, absence of ICD band, indicated the external groove binding/binding somewhere at loops. This is also evident in molecular docking results, the ligand binds to groove forming base (G4, G5, G24, T25) and in the vicinity to TTA loop (G14, G15, T17) bases of wHTel26 and HTel22, respectively. Thermal stabilization induced by ligand was found greater in Na+ ion (27.5 °C) than (19.1 °C) in K+ ion. Ligand caused cell toxicity in MCF-7 cancer cell lines with an IC50 value of ∼8.4 µM. The above findings suggest the ligand, 1,5-bis[3-(diethylamino)propionamido]anthracene-9,10-dione could be a potent anticancer drug candidate and has great therapeutic implications. Binding of disubstituted amido anthraquinone derivative, 1,5-bis[3-(diethylamino)propionamido]anthracene-9,10-dione to human telomere HTel22 antiparallel conformation induced thermal stabilization. Communicated by Ramaswamy H. Sarma

The mechanism of UP1 binding and unfolding of human telomeric DNA G-quadruplex

The human telomere contains multiple copies of the DNA sequence d(TTAGGG) which can fold into higher order intramolecular G-quadruplexes and regulate the maintenance of telomere length and chromosomal integrity. The nucleic acid binding protein heteronuclear ribonucleoprotein A1 (hnRNP A1) and its N-terminus proteolytic product UP1 have been shown to efficiently bind and unfold telomeric DNA G-quadruplex. However, the understanding of the molecular mechanism of the UP1 binding and unfolding telomeric G-quadruplexes is still limited. Here, we performed biochemical and biophysical characterizations of UP1 binding and unfolding of human telomeric DNA G-quadruplex d[AGGG(TTAGGG)3], and in combination of systematic site-direct mutagenesis of two tandem RNA recognition motifs (RRMs) in UP1, revealed that RRM1 is responsible for initial binding and unfolding, whereas RRM2 assists RRM1 to complete the unfolding of G-quadruplex. Isothermal titration calorimetry (ITC) and circular dichroism (CD) studies of the interactions between UP1 and DNA G-quadruplex variants indicate that the “TAG” binding motif in Loop2 of telomeric G-quadruplex is critical for UP1 recognition and G-quadruplex unfolding initiation. Together we depict a model for molecular mechanism of hnRNP A1 (UP1) binding and unfolding of the human telomeric DNA G-quadruplex.

Use of Single-Molecule Plasmon-Enhanced Fluorescence to Investigate Ligand Binding to G-Quadruplex DNA.

Single-molecule measurements are crucial for studying the interactions between G-quadruplex (GQ) DNA and ligands, as they provide higher resolution and sensitivity compared to those of bulk measurements. In this study, we employed plasmon-enhanced fluorescence to investigate the real-time interaction between the cationic porphyrin ligand TmPyP4 and different topologies of telomeric GQ DNA at the single-molecule level. By analyzing the time traces of the fluorescence bursts, we extracted dwell times for the ligand. For parallel telomeric GQ DNA, the dwell time distribution followed a biexponential fit, yielding mean dwell times of 5.6 and 18.6 ms. For the antiparallel topology of human telomeric GQ DNA, plasmon-enhanced fluorescence of TmPyP4 was observed, with dwell time distributions following a single-exponential fit and a mean dwell time of 5.9 ms. Our approach allows the nuances of GQ-ligand interactions to be captured and holds promise for studying weakly emitting GQ ligands at the single-molecule level.

Crystal structure of a three-tetrad, parallel, K+-stabilized human telomeric G-quadruplex at 1.35 Å resolution.

The crystal structure of the G-rich human telomeric DNA Tel22 has been determined at 1.35 Å resolution in space group P6. Tel22 forms a non-canonical DNA structure called the G-quadruplex. The space group and unit-cell parameters are comparable to those in the crystal structures with PDB codes 6ip3 (1.40 Å resolution) and 1kf1 (2.15 Å resolution). The G-quadruplexes are highly similar in all of the structures. However, this structure of Tel22 displays clear density for polyethylene glycol and two potassium ions, which are located outside the ion channel in the G-quadruplex and play an important role in stabilizing the crystal contacts. In addition, 111 water molecules were identified (compared with 79 and 68 in PDB entries 6ip3 and 1kf1, respectively) that participate in intricate and extensive networks providing high stability to the G-quadruplex.

Stability of Human Telomeric G-Quadruplexes Complexed with Photosensitive Ligands and Irradiated with Visible Light.

Guanine-rich DNA sequences can fold into non-canonical nucleic acid structures called G-quadruplexes (G4s). These nanostructures have strong implications in many fields, from medical science to bottom-up nanotechnologies. As a result, ligands interacting with G4s have attracted great attention as candidates in medical therapies, molecular probe applications, and biosensing. In recent years, the use of G4-ligand complexes as photopharmacological targets has shown significant promise for developing novel therapeutic strategies and nanodevices. Here, we studied the possibility of manipulating the secondary structure of a human telomeric G4 sequence through the interaction with two photosensitive ligands, DTE and TMPyP4, whose response to visible light is different. The effect of these two ligands on G4 thermal unfolding was also considered, revealing the occurrence of peculiar multi-step melting pathways and the different attitudes of the two molecules on the quadruplex stabilization.

Reversible Photo-switching of G‐Quadruplex by Backbone-Inserted Azobenzene

Introduction: Taking the typical human telomeric G-quadruplex (G4) sequence as a reference, we designed four photoresponsive DNA sequences by inserting azobenzene moieties into a planar interlayer and the end surfaces of the G4 structure. Method: The photo-responsive G4 molecules were investigated by melting curve, FRET, CD, and gel electrophoresis. Results and Discussion: The measurements showed that the photo-responsive G4 molecules took the stable four-strand structure under visible light, but after UV light irradiation the G4 structures tended to be disentangled. When azobenzene molecules were inserted at the end surfaces of the G4 structure, the Tm difference of the photo-responsive G4 between visible light and UV light reached more than 30 oC. Conclusion: In a temperature range from 20 to 53 oC, the reversible transformation of the G4 structure can be realized solely by light irradiation.

Inhibited complete folding of consecutive human telomeric G-quadruplexes.

Noncanonical DNA structures, termed G-quadruplexes, are present in human genomic DNA and are important elements in many DNA metabolic processes. Multiple sites in the human genome have G-rich DNA stretches able to support formation of several consecutive G-quadruplexes. One of those sites is the telomeric overhang region that has multiple repeats of TTAGGG and is tightly associated with both cancer and aging. We investigated the folding of consecutive G-quadruplexes in both potassium- and sodium-containing solutions using single-molecule FRET spectroscopy, circular dichroism, thermal melting and molecular dynamics simulations. Our observations show coexistence of partially and fully folded DNA, the latter consisting of consecutive G-quadruplexes. Following the folding process over hours in sodium-containing buffers revealed fast G-quadruplex folding but slow establishment of thermodynamic equilibrium. We find that full consecutive G-quadruplex formation is inhibited by the many DNA structures randomly nucleating on the DNA, some of which are off-path conformations that need to unfold to allow full folding. Our study allows describing consecutive G-quadruplex formation in both nonequilibrium and equilibrium conditions by a unified picture, where, due to the many possible DNA conformations, full folding with consecutive G-quadruplexes as beads on a string is not necessarily achieved.

Quantum tunneling time delay investigation of [Formula: see text] ion in human telomeric G-quadruplex systems.

Guanine-rich quadruplex DNA (G-quadruplex) is of interest both in cell biology and nanotechnology. Its biological functions necessitate a G-quadruplex to be stabilized against escape of the monovalent metal cations. The potassium ion ([Formula: see text]) is particularly important as it experiences a potential energy barrier while it enters and exits the G-quadruplex systems which are normally found in human telomere. In the present work, we analyzed the time it takes for the [Formula: see text] cations to get in and out of the G-quadruplex. Our time estimate is based on entropic tunneling time-a time formula which gave biologically relevant results for DNA point mutation by proton tunneling. The potential energy barrier experienced by [Formula: see text] ions is determined from a quantum mechanical simulation study, Schrodinger equation is solved using MATLAB, and the computed eigenfunctions and eigenenergies are used in the entropic tunneling time formula to compute the time delay and charge accumulation rate during the tunneling of [Formula: see text] in G-quadruplex. The computations have shown that ion tunneling takes picosecond times. In addition, average [Formula: see text] accumulation rate is found to be in the picoampere range. Our results show that time delay during the [Formula: see text] ion tunneling is in the ballpark of the conformational transition times in biological systems, and it could be an important parameter for understanding its biological role in human DNA as well as for the possible applications in biotechnology. To our knowledge, for the first time in the literature, time delay during the ion tunneling from and into G-quadruplexes is computed.

The anticancer peptide LL-III binds with nanomolar affinity to human telomeric and cMyc G-quadruplexes.

LL-III is a natural anticancer peptide able to cross the membrane of cancer cells and to localize in the nucleolus, but its intracellular target is unknown. Here, we show that LL-III is able to bind with nM affinity to specific G-quadruplex structures known to be relevant anticancer targets.

Revealing light-induced structural shifts in G-quadruplex-porphyrin complexes: a pulsed dipolar EPR study.

The binding of G-quadruplex structures (G4s) with photosensitizers is of considerable importance in medicinal chemistry and drug discovery due to their promising potential in photodynamic therapy applications. G4s can experience structural changes as a result of ligand interactions and light exposure. Understanding these modifications is essential to uncover the fundamental biological roles of the complexes and optimize their therapeutic potential. The structural diversity of G4s makes it challenging to study their complexes with ligands, necessitating the use of various complementary methods to fully understand these interactions. In this study, we introduce, for the first time, the application of laser-induced dipolar EPR as a method to characterize G-quadruplex DNA complexes containing photosensitizers and to investigate light-induced structural modifications in these systems. To demonstrate the feasibility of this approach, we studied complexes of the human telomeric G-quadruplex (HTel-22) with cationic 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphyrin tetra(p-toluenesulfonate) (TMPyP4). In addition to showcasing a new methodology, we also aimed to provide insights into the mechanisms underlying photoinduced HTel-22/TMPyP4 structural changes, thereby aiding in the advancement of approaches targeting G4s in photodynamic therapy. EPR revealed G-quadruplex unfolding and dimer formation upon light exposure. Our findings demonstrate the potential of EPR spectroscopy for examining G4 complexes with photosensitizers and contribute to a better understanding of G4s' interactions with ligands under light.