Human Telomeric Sequence Research Articles

Protein condensates unfold G-quadruplex resembling a helicase activity.

Membrane-less organelles, formed by liquid-liquid phase separation, participate in many vital cellular processes and have received extensive attention recently. A notable form of noncanonical nucleic acid secondary structure, G-quadruplex (G4), interacts with the scaffolding proteins in these membrane-less organelles and becomes an integral part of this condensed phase. However, the structure and stability features of the integrated G4 remain poorly characterized. Herein, we employed NMR along with other biophysical methods to investigate the conformation of a G4 within condensates formed by a disordered protein known as DDX4N1. We discovered that the human telomeric sequence MHT24, which forms a G4 structure in a non-condensed phase solution of protein DDX4N1, unfolds when it is within DDX4N1 condensates due to phase separation. Our findings provide an instance of a protein acquiring new functionality through phase separation process, which deepen our understanding of how protein condensates regulate G4 structure and their functions.

Molecular Docking Study of Binding of Perylene Di-imide to a Bio Molecular Human Telomeric G-quadruplex

Human telomeres are comprised of d(TTAGGG) repeats involved in the formation of G-quadruplex DNA structures. Ligands stabilizing these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. In human cells , telomerase adds multiple copies of the 5’-GGTTAG-3’ motif to the end of the G-strand of the telomere and in the majority of tumor cells it results over-expressed. Several structural studies have revealed a diversity of topologies for telomeric quadruplexes, which are sensitive to the nature of the cations present, to the flanking sequences, and probably also to concentration, as confirmed by the different conformations deposited in the Protein Data Bank (PDB). The existence of different polymorphism in the DNA quadruplex and the absence of a uniquely precise binding site give rise to check docking approach . As target we have selected six different experimental models of the human telomeric sequence d[AG3(T2AG3)3] based on three G-tetrads and as ligands the perylene di-imide . We checked out molecular docking simulation of binding of perylene di-imide to a slected G-quadruplex using dock 6.9 to examine whether or not to reproduced the loop binding mode of perylene di-imide. The simulation gave the two highest rank docking pose of perylene di-imide and the binding mode were external stacking on the terminal guanine tetrade and the groove binding.

A pH-Responsive Topological Switch Based on a DNA Quadruplex-Duplex Hybrid.

A guanine-rich oligonucleotide based on a human telomeric sequence but with the first three-nucleotide intervening stretch replaced by a putative 15-nucleotide hairpin-forming sequence shows a pH-dependent folding into different quadruplex-duplex hybrids in a potassium containing buffer. At slightly acidic pH, the quadruplex domain adopts a chair-type conformation. Upon increasing the pH, a transition with a midpoint close to neutral pH to a major and minor (3+1) hybrid topology with either a coaxially stacked or orthogonally oriented duplex stem-loop occurs. NMR-derived high-resolution structures reveal that an adenine protonation is prerequisite for the formation of a non-canonical base quartet, capping the outer G-tetrad at the quadruplex-duplex interface and stabilizing the antiparallel chair conformation in an acidic environment. Being directly associated with interactions at the quadruplex-duplex interface, this unique pH-dependent topological transition is fully reversible. Coupled with a conformation-sensitive optical readout demonstrated as a proof of concept using the fluorescent dye thiazole orange, the present quadruplex-duplex hybrid architecture represents a potentially valuable pH-sensing system responsive in a physiological pH range of 7±1.

Structural Differences at Quadruplex-Duplex Interfaces Enable Ligand-Induced Topological Transitions.

Quadruplex-duplex (QD) junctions, which represent unique structural motifs of both biological and technological significance, have been shown to constitute high-affinity binding sites for various ligands. A QD hybrid construct based on a human telomeric sequence, which harbors a duplex stem-loop in place of a short lateral loop, is structurally characterized by NMR. It folds into two major species with a (3+1) hybrid and a chair-type (2+2) antiparallel quadruplex domain coexisting in a K+ buffer solution. The antiparallel species is stabilized by an unusual capping structure involving a thymine and protonated adenine base AH+ of the lateral loop facing the hairpin duplex to form a T·AH+·G·C quartet with the interfacial G·C base pair at neutral pH. Addition and binding of Phen-DC3 to the QD hybrid mixture by its partial intercalation at corresponding QD junctions leads to a topological transition with exclusive formation of the (3+1) hybrid fold. In agreement with the available experimental data, such an unprecedented discrimination of QD junctions by a ligand can be rationalized following an induced fit mechanism.

Mismatch repair enzymes regulate telomere recombination in Saccharomycescerevisiae

DNA mismatch repair (MMR) is a crucial mechanism that ensures chromosome stability and prevents the development of various human cancers. Apart from its role in correcting mismatches during DNA replication, MMR also plays a significant role in regulating recombination between non-identical sequences, a process known as homeologous recombination. Telomeres, the protective ends of eukaryotic chromosomes, possess sequences that are not perfectly homologous. While telomerase primarily maintains telomere length in the yeast Saccharomyces cerevisiae, recombination between telomeres becomes a major pathway for length maintenance in cells lacking telomerase. This study investigates the participation of MMR in telomere recombination. Our findings reveal that mutations in MMR genes activate type I recombination. Notably, among the MMR proteins, MutSα (Msh2 and Msh6) and MutLα (Mlh1 and Pms1) exerted the most pronounced effects on telomere recombination. We also found that yeast cells containing simple human telomeric TTAGGG DNA sequences preferentially utilize type II recombination to maintain their telomeres, highlighting the influence of the heterogeneous nature of yeast telomeric sequences on type II recombination. Furthermore, our observations indicate that MMR activity is indispensable for its impact on telomere recombination. Collectively, these results contribute to a more comprehensive understanding of the role of MMR in telomere recombination.

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 .

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.

Basic protein- and peptide-induced stabilization of long-loop DNA G-guadruplexes

The human genome contains many G-quadruplex-forming sequences, including sequences containing long single-stranded loops that are believed to be unfavorable for G-quadruplex formation. The intracellular environment of biological cells is crowded with proteins with charged surfaces. Understanding the effects of protein-rich environments is important for understanding the formation of G-quadruplexes in an intracellular environment. In this study, we investigated the structural stability of DNA G-quadruplexes in the presence of several types of globular proteins (lysozyme, cytochrome c, bovine serum albumin, myoglobin, histone proteins, and serum proteins), unstructured polypeptides (protamine and poly-l-lysine), and oligopeptides (RGG/RG-domain peptides and short repeated peptides). Thermal melting studies of G-quadruplex-forming oligonucleotides derived from the human telomeric repeat sequence revealed that environments containing high concentrations of proteins and peptides differently affected the G-quadruplex stability according to their loop lengths. We found that weak electrostatic interactions of G-quadruplex loops with basic proteins and peptides improved the stability of long-loop G-quadruplexes and the interactions were strengthened under crowded conditions simulated by dextran. The comparison of the effects of different types of proteins and peptides indicated that excluded volume interactions and structural flexibility of both DNA and polypeptide chains influenced the efficiency of their interactions. This study provides insights into long-loop G-quadruplex stability in a crowded intracellular environment and the recognition of G-quadruplexes by arginine-rich domains of G-quadruplex-binding proteins.

Light rare earth elements stabilize G-quadruplex structure in variants of human telomeric sequences

Recently, light rare earth elements (LREEs) are gaining importance in modern-day technologies. Thus, the entry of LREEs into biochemical pathways cannot be ignored, which might affect the conformation of biomacromolecules. Herein, for the first time, we discover the G-quadruplex formation in the human telomeric variants in presence of micromolar concentrations of LREEs. Thermal melting show that the LREE-induced unimolecular G-quadruplex structure. Isothermal titration calorimetry, UV–vis, and CD spectroscopy results suggest the binding stoichiometry of lanthanide ions to telomeric variants is 2:1. The data confirms that the LREE ions coordinate between adjacent G-quartets. The excess LREE ions are most likely binding to quadruplex loops. The CD spectra revealed that the LREE-induced quadruplex in human telomere and its variant have antiparallel orientation. The binding equilibria of LREEs have been studied both in the presence and absence of competing metal cations. Addition of LREEs to the Na+ or K+-induced G-quadruplexes led to conformational change, which may be ascribed to the displacement of K+ or Na+ ions by LREE ions and formation of a more compact LREE-induced G-quadruplex structure in human telomeric variant. Moreover, the thymine in the central loop of the human telomeric sequence stabilizes LREE induced G-quadruplex.

Computational Probing of the Folding Mechanism of Human Telomeric G-Quadruplex DNA.

The human telomeric (htel) sequences in the terminal regions of human telomeres form diverse G-quadruplex (GQ) structures. Despite much experimental efforts to elucidate the folding pathways of htel GQ, no comprehensive model of htel GQ folding has been presented. Here, we describe folding pathways of the htel GQ determined by state-of-the-art enhanced sampling molecular dynamics simulation at the all-atom level. Briefly, GQ folding is initiated by the formation of a single-hairpin and then followed by the formation of double-hairpins, which then branch via distinct folding pathways to produce different GQ topologies (antiparallel chair, antiparallel basket, hybrids 1 and 2, and parallel propeller). In addition to these double-hairpin states, three-triad and two-tetrad structures in antiparallel backbone alignment serve as key intermediates that connect the GQ folding and transition between two different GQs.

Spectroscopy evidence of interaction of 1,5 disubstituted piperidino-amido anthraquinone derivative with human telomeric G-quadruplex DNA: Basis of anticancer action

The binding of ligands to G-quadruplex (G4) structures at the ends of human telomeric DNA induces thermal stabilization, which interferes with telomere maintenance by disrupting the association of telomeres with the telomerase enzyme—an important marker for cancer. Understanding the binding mode of the ligand-G quadruplex DNA complex is imperative for evaluating the relative efficacy and specificity of their interaction. We focused on the interaction of 1,5-Bis(3-piperidino propionamido) anthracene-9,10-dione with human telomeric DNA sequences using surface plasmon resonance, absorbance, fluorescence (steady-state and lifetime), and circular dichroism spectroscopy techniques. The ligand binding with HTel22 (Kb = 8.4 × 105 M−1) induced cell toxicity with an IC50 value of ∼ 8.64 µM in MCF-7 cancer cell lines. Significant hypochromism (59 %), fluorescence quenching (97 %), no change in fluorescence lifetime and absence of induced Circular Dichroism (CD) band upon addition of G4 DNA to ligand, suggest a groove/external binding mode. CD spectral changes reflect rearrangement in parallel and antiparallel strands to accommodate the ligand. Docking results reveal specific short contacts of the ligand's side chain, including 9CO, 13N, 14NH, and 11CO, with the grooves of the G4 DNA, without making any contact with the loops, favoring an energetically more favorable conformation. Thermal denaturation profiles, obtained by Differential Scanning Calorimetry and CD, show the stabilization of wHTel26 and HTel22 G4 DNA in K+ and Na+ rich solutions by 21.2 and 17.6 °C, respectively, which may restrict the access of telomerase to telomeres. The findings indicate the potential of modifying anthraquinone substituent groups for therapeutic applications.

Prospecting the cancer therapeutic edge of chitosan-based gold nanoparticles through conformation selective binding to the parallel G-quadruplex formed by short telomeric DNA sequence: A multi-spectroscopic approach

The biological relevance of G4 structures formed in telomere & oncogenes promoters make them extremely crucial therapeutic target for cancer treatment. Herein, we have synthesized chitosan-based gold nanoparticles (CH-Au NPs) through green method and have investigated their interaction with G4 structures formed by short telomeric sequences to evaluate their potential for targeting G4 structures. Firstly, we have characterized morphological/physical attributes of synthesized CH-Au NPs and salt dependent structural aspects of model G-rich DNA sequence, 12-mer d(T2G4)2 [TETRA] using spectroscopic and biophysical techniques. The molecular interactions between CH-Au NPs and parallel/antiparallel TETRA G4 structures were evaluated using UV–Visible, CD, Fluorescence, CD melting, DLS and Zeta potential studies. The experimental data indicated that CH-Au NPs showed strong binding interactions with Parallel TETRA G4 and provided thermal stabilization to the structure, whereas their interactions with Antiparallel TETRA G4 DNA and Ct-DNA (DNA duplex) were found to be negligible. Further, CH-Au NPs were also investigated for their selectivity aptitude for different G4 structures formed by human telomeric sequences; d(T2AG3)3 [HUM-12] and d(T2AG3)4T [HUM-25]. Our findings suggested that CH-Au NPs exhibited topology specific binding aptitude towards G4 structure, which can be utilized to inhibit/modulate crucial biological functions for potential anticancer activity.

Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.

G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

A ThT Derivative as Zn2+ Sensor Based on DNA G-quadruplex.

In this report, we developed a sensing strategy based on ThT-E(a ThT derivative) and DNA G-quadruplex for the label-free detection of Zn2+. In the absence of Zn2+, there was a fluorescence enhancement of ThT-E by interaction with human telomere sequence. On the addition of Zn2+, Zn2+ induced a more compact antiparallel G-quadruplex to release ThT-E, resulting in fluorescence quenching. The detection limit was 0.6996μM, and the fluorescence intensity showed a good linear relationship with the concentration of Zn2+ in the range of 0-10μM. This sensing strategy which only needs to mix two kinds of materials has the characteristics of label-feel, simple operation, short response time, economical and efficient.

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.

A Bright and Ionizable Cytosine Mimic for i-Motif Structures.

A new fluorescent cytosine analog "tsC" containing a trans-stilbene moiety was synthesized and incorporated into hemiprotonated base pairs that comprise i-motif structures. Unlike previously reported fluorescent base analogs, tsC mimics the acid-base properties of cytosine (pKa ≈ 4.3) while exhibiting bright (ε × Φ ≈ 1000 cm-1 M-1) and red-shifted fluorescence (λem = 440 → 490 nm) upon its protonation in the water-excluded interface of tsC+:C base pairs. Ratiometric analyses of tsC emission wavelengths facilitate real-time tracking of reversible conversions between single-stranded, double-stranded, and i-motif structures derived from the human telomeric repeat sequence. Comparisons between local changes in tsC protonation with global structure changes according to circular dichroism suggest partial formation of hemiprotonated base pairs in the absence of global i-motif structures at pH = 6.0. In addition to providing a highly fluorescent and ionizable cytosine analog, these results suggest that hemiprotonated C+:C base pairs can form in partially folded single-stranded DNA in the absence of global i-motif structures.

Asymmetric triplex metallohelices stabilise DNA G-quadruplexes in promoter oncogene sequences and efficiently reduce their expression in cancer cells

Some metallo-supramolecular helical assemblies with size, shape, charge and amphipathic architectures similar to short cationic α-helical peptides have been shown to target and stabilise DNA G-quadruplexes (G4s) in vitro and downregulate the expression of G4-regulated genes in human cells. To expand the library of metallohelical structures that can act as efficient DNA G4 binders and downregulate genes containing G4-forming sequences in their promoter regions, we investigated the interaction of the two enantiomeric pairs of asymmetric Fe(II) triplex metallohelices with a series of five different DNA G4s formed by the human telomeric sequence (hTelo) and in the promoter regions of c-MYC, c-KIT, and k-RAS oncogenes. The metallohelices display preferential binding to G4s over duplex DNA in all investigated G4-forming sequences and induced arrest of DNA polymerase on template strands containing G4-forming sequences. Moreover, the investigated metallohelices suppressed the expression of c-MYC and k-RAS genes at mRNA and protein levels in HCT116 human cancer cells, as revealed by RT-qPCR analysis and western blotting.

Two Opposing Effects of Monovalent Cations on the Stability of i-Motif Structure.

At acidic pH, cytosine-rich single-stranded DNA can be folded into a tetraplex structure called i-motif (iM). In recent studies, the effect of monovalent cations on the stability of iM structure has been addressed, but a consensus about the issue has not been reached yet. Thus, we investigated the effects of various factors on the stability of iM structure using fluorescence resonance energy transfer (FRET)-based analysis for three types of iM derived from human telomere sequences. We confirmed that the protonated cytosine-cytosine (C:C+) base pair is destabilized as the concentration of monovalent cations (Li+, Na+, K+) increases and that Li+ has the greatest tendency of destabilization. Intriguingly, monovalent cations would play an ambivalent role in iM formation by making single-stranded DNA flexible and pliant for an iM structure. In particular, we found that Li+ has a notably greater flexibilizing effect than Na+ and K+. All taken together, we conclude that the stability of iM structure is controlled by the subtle balance of the two counteractive effects of monovalent cations: electrostatic screening and disruption of cytosine base pairing.

Computing the electronic circular dichroism spectrum of DNA quadruple helices of different topology: A critical test for a generalized excitonic model based on a fragment diabatization.

In this study, we exploit a recently developed fragment diabatization-based excitonic model, FrDEx, to simulate the electronic circular dichroism (ECD) spectra of three guanine-rich DNA sequences arranged in guanine quadruple helices with different topologies: thrombin binding aptamer (antiparallel), c-Myc promoter (parallel), and human telomeric sequence (3+1 hybrid). Starting from time-dependent density functional theory (TD-DFT) calculations with the M052X functional, we apply our protocol to parameterize the FrDEX Hamiltonian, which accounts for electron density overlap and includes both the coupling with charge transfer transitions and the effect of the surrounding bases on the local excitation of each chromophore. The TD-DFT/M052X spectral shapes are in good agreement with the experimental ones, the main source of discrepancy being related to the intrinsic error on the computed transition energies of guanine monomer. FrDEx spectra are fairly close to the reference TD-DFT ones, allowing a significant advance with respect to a more standard excitonic Hamiltonian. We also show that the ECD spectra are sensitive to the inclusion of the inner K cation in the calculation.

Luminescent Ruthenium(II) Complexes Used for the Detection of 8-Oxoguanine in the Human Telomeric Sequence.

Detecting cancer at the early stage of the disease is crucial to keep the best chance for successful treatment. The recent development of genomic screening, a methodology that is addressed to asymptomatic patients presumably at risk of carcinogenesis, has stimulated the quest for new tools able to signal the level of risk. Carcinogenesis has been associated to chronic oxidative stress exceeding the antioxidant defenses and leading to critical genome alteration levels. The telomeric regions are presumably the most exposed to oxidative stress due to their high concentration of guanine (i.e., the easiest oxidizable nucleic base). Accumulation of 8-oxoguanine in telomeres, thus oxidative lesions, was reportedly associated with telomeric crisis and carcinogenesis. In this study, we report on the capacity of Ru(II) polyazaaromatic complexes to photoprobe 8-oxoguanine into the human telomeric sequence with the view of developing new tools for cancer risk screening.