Guanine-rich DNA Sequences Research Articles

Self-priming dumbbell probe-mediated cascade isothermal amplification for sensitive and label-free MICRORNA detection using fluorescence light-up silver nanocluster

Given the critical significance of microRNAs (miRNAs) in cancer diagnosis and classification (e.g., glioma), there is a need for the advancement of sensitive, specific, quantitative, and cost-effective approach for assessing miRNA expression levels. We present an effective amplification technology for ultrasensitive miRNA analysis by combining a self-priming dumbbell probe-mediated cascade isothermal amplification strategy with fluorescence-enhanced silver nanoclusters (DNA-AgNCs). The approach depends on the disassociation of the stem portion of the dumbbell probe through the binding of target miRNA, followed by self-priming mediated target recycling, DNA polymerase/endonuclease facilitated chain extension, and catalytic hairpin assembly (CHA) to provide an amplified signal. The CHA procedure generates an LP-AgNCs/LP-G duplex, wherein the fluorescence signals of DNA-AgNCs can be significantly amplified when in close proximity to guanine-rich (G-rich) DNA sequences. The proposed label-free assay, characterized by high sensitivity, a wide dynamic range (spanning 6 orders of magnitude), excellent specificity (capable of detecting single-base differences), and simple operation, has been successfully utilized for the detection of miRNA in real samples. This demonstrates its potential as a compelling alternative for miRNA analysis in gene expression profiling and molecular diagnostics, especially for early glioma cancer detection.

Differential binding of curcumin with chair and basket type anti-parallel DNA G-quadruplexes

Guanine rich DNA sequences are widespread throughout the genome and they are prone to form G-quadruplex structures both in-vitro and in-vivo. Owing to their regulatory roles in important biological reactions, DNA G-quadruplexes are the prime target for the development of anti-cancer drug molecules. In the present study, we have investigated the interaction of curcumin with anti-parallel chair and basket type DNA G-quadruplexes formed by thrombin binding aptamer (TBA) and human telomeric repeat (HTG) sequences, respectively. The results obtained from spectroscopic techniques demonstrated that curcumin interacts with both the DNA G-quadruplexes through external binding mode and the estimated apparent binding constant value (Kb) was slightly higher for HTG (Kb = (4.38 ± 0.09) × 105 M−1) than that estimated for TBA (Kb = (0.62 ± 0.02) × 105 M−1) DNA G-quadruplex structures. It was also revealed that upon binding with curcumin, TBA and HTG DNA G-quadruplexes were slightly stabilized and destabilized, respectively. Circular dichroism study revealed that upon binding with curcumin the global structure of both the DNA G-quadruplexes was unaltered, whereas the local structure of HTG DNA G-quaruplex was slightly altered. Conjointly all the aforementioned spectroscopic methods emphasize that curcumin has differential interaction with anti-parallel DNA G-quadruplex structures depending on their loop orientation.

Research progress in targeting DNA G-quadruplexes using natural products

DNA G-quadruplex(G4) is a guanine-rich single-stranded DNA sequence that spontaneously folds into a spherical four-stranded DNA secondary structure in oncogene promoter sequences and telomeres. G4s are highly associated with the occurrence and development of cancer and have emerged as promising anticancer targets. Natural products have long been important sources of anticancer drug development. In recent years, significant progress has been made in the discovery of natural drugs targeting DNA G4s, with many DNA G4s have been confirmed as promising targets of natural products, including MYC-G4, KRAS-G4, PDGFR-β-G4, BCL-2-G4, VEGF-G4, and telomeric G4. This review summarizes the research progress in discovering natural small molecules that target DNA G4s and their binding mechanisms. It also discusses the opportunities of and challenges in developing drugs targeting DNA G4s. This review will serve as a valuable reference for the research on natural products, particularly in the development of novel antitumor medications.

Structural Basis for Parallel G-Quadruplex Recognition by an Ankyrin Protein.

G-Quadruplex (G4) structures formed by guanine-rich DNA and RNA sequences are implicated in various biological processes. Understanding the mechanisms by which proteins recognize G4 structures is crucial for elucidating their functional roles. Here we present the X-ray crystal structure of an ankyrin protein bound to a parallel G4 structure. Our findings reveal a new specific recognition mode in which a bundle of α-helices and loops of the ankyrin form a flat surface to stack on the G-tetrad core. The protein employs a combination of hydrogen bonds and hydrophobic contacts to interact with the G4, and electrostatic interaction is used to enhance the binding affinity. This binding mechanism provides valuable insights into understanding G4 recognition by proteins.

Targeting G4 motifs of various stem cell makers with designed peptide for therapeutic applications

Noncanonical secondary structures formed by Guanine-rich DNA sequences fold into four-stranded structures called the G-quadruplexes (G4s). Targeting G-quadruplexes is considered an attractive approach toward drug intervention. Here, we have studied the targeting of G4s of stem cell markers with designed short peptide (named as QW10) using biophysical and biochemical techniques. Our CD studies showed that G4 sequences of stem cell markers formed mixed G-quadruplexes in 100 mM Na+, 100 mM K+ and 100 mM K+ +40 wt% PEG 200. On titrating these structures with an increasing concentration of QW10 peptide, we observed a significant decrease in CD intensity followed by the complete disappearance of G4 CD signatures confirming their destabilization not only in dilute conditions but also under cell-mimicking molecular crowding conditions. Our electrophoretic mobility shift assay and significant decrease in the Tm values confirmed the significant destabilization of G4 structures Fluorescence results showed the formation of high-affinity G4 complex-peptide complex with binding affinities in the micromolar (µM) range of 2–8 µM in different ionic conditions. First time, this study may give insight into the use of peptides as leads for the development of more potent and selective ligands to regulate the potential therapeutic applications of cancer stem cell markers.

Cell redistribution of G quadruplex-structured DNA is associated with morphological changes of nuclei and nucleoli in neurons during tau pathology progression.

While the double helical structure has long been its iconic representation, DNA is structurally dynamic and can adopt alternative secondary configurations. Specifically, guanine-rich DNA sequences can fold in guanine quadruplexes (G4) structures. These G4 play pivotal roles as regulators of gene expression and genomic stability, and influence protein homeostasis. Despite their significance, the association of G4 with neurodegenerative diseases such as Alzheimer's disease (AD) has been underappreciated. Recent findings have identified DNA sequences predicted to form G4 in sarkosyl-insoluble aggregates from AD brains, questioning the involvement of G4-structured DNA (G4 DNA) in the pathology. Using immunofluorescence coupled to confocal microscopy analysis we investigated the impact of tau pathology, a hallmark of tauopathies including AD, on the distribution of G4 DNA in murine neurons and its relevance to AD brains. In healthy neurons, G4 DNA is detected in nuclei with a notable presence in nucleoli. However, in a transgenic mouse model of tau pathology (THY-Tau22), early stages of the disease exhibit an impairment in the nuclear distribution of G4 DNA. In addition, G4 DNA accumulates in the cytoplasm of neurons exhibiting oligomerized tau and oxidative DNA damage. This altered distribution persists in the later stage of the pathology when larger tau aggregates are present. Still cytoplasmic deposition of G4 DNA does not appear to be a critical factor in the tau aggregation process. Similar patterns are observed in neurons from the AD cortex. Furthermore, the disturbance in G4 DNA distribution is associated with various changes in the size of neuronal nuclei and nucleoli, indicative of responses to stress and the activation of pro-survival mechanisms. Our results shed light on a significant impact of tau pathology on the dynamics of G4 DNA and on nuclear and nucleolar mechanobiology in neurons. These findings reveal new dimensions in the etiopathogenesis of tauopathies.

Mechanical Stability and Unfolding Pathways of Parallel Tetrameric G-Quadruplexes Probed by Pulling Simulations.

Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects remain elusive, despite being important for understanding structure formation and biological functions of GQs. Pulling experiments are a common tool for acquiring insights into the folding landscape of GQs. Herein, we applied a computational pulling strategy─steered molecular dynamics (SMD) simulations─in combination with standard molecular dynamics (MD) simulations to explore the unfolding landscapes of tetrameric parallel GQs. We identified anisotropic properties of elastic conformational changes, unfolding transitions, and GQ mechanical stabilities. Using a special set of structural parameters, we found that the vertical component of pulling force (perpendicular to the average G-quartet plane) plays a significant role in disrupting GQ structures and weakening their mechanical stabilities. We demonstrated that the magnitude of the vertical force component depends on the pulling anchor positions and the number of G-quartets. Typical unfolding transitions for tetrameric parallel GQs involve base unzipping, opening of the G-stem, strand slippage, and rotation to cross-like structures. The unzipping was detected as the first and dominant unfolding event, and it usually started at the 3'-end. Furthermore, results from both SMD and standard MD simulations indicate that partial spiral conformations serve as a transient ensemble during the (un)folding of GQs.

Comprehensive analysis of intramolecular G-quadruplex structures: furthering the understanding of their formalism.

G-quadruplexes (G4) are helical structures found in guanine-rich DNA or RNA sequences. Generally, their formalism is based on a few dozen structures, which can produce some inconsistencies or incompleteness. Using the website ASC-G4, we analyzed the structures of 333 intramolecular G4s, of all types, which allowed us to clarify some key concepts and present new information. To each of the eight distinguishable topologies corresponds a groove-width signature and a predominant glycosidic configuration (gc) pattern governed by the directions of the strands. The relative orientations of the stacking guanines within the strands, which we quantified and related to their vertical gc successions, determine the twist and tilt of the helices. The latter impact the minimum groove widths, which represent the space available for lateral ligand binding. The G4 four helices have similar twists, even when these twists are irregular, meaning that they have various angles along the strands. Despite its importance, the vertical gc succession has no strict one-to-one relationship with the topology, which explains the discrepancy between some topologies and their corresponding circular dichroism spectra. This study allowed us to introduce the new concept of platypus G4s, which are structures with properties corresponding to several topologies.

Organic-soluble cytosine derivatives for studying base-pair interactions in nonaqueous solution

The presence of higher order G-quadruplex-DNA (G4) structures in human cells in guanine-rich DNA sequences has been well established. In cases of G4 helicase impairment or G4 stabilization via association with G4 ligands, it has been determined that arrest and/or termination of DNA transcription/replication occurs because these structures act as physical barriers to both helicase and polymerase mobility along the DNA. Truncation of transcription or replication may result in the loss of genetic material expression leading to disorders. To ameliorate G4-induced barriers, agents that destabilize or “unwind” the quadraplex structures are sought. Earlier work has shown that the cytosine analogue phenylpyrrolocytosine has potential G4-destabilizing ability. It was hypothesized that the unwinding activity was related to its ability to base pair with guanosine. To facilitate the design and evaluation of the hydrogen bonding ability of cytosine analogues, solubility in aprotic, and low-polarity solvents, e.g., deutero-chloroform (CDCl3), is needed; then, determination of association constants ( Ka) with guanine via 1H nuclear magnetic resonance (NMR) spectroscopy or isothermal titration calorimetry (ITC) will be possible. Herein, we report on the synthesis of three new chloroform-soluble, cytosine analogues: N1-cyclohexylmethyl-7-phenylimidazolocytosine, N1-cyclohexylmethyl-7-(6-(methoxycarbonyl)pyridin-2-yl)imidazolocytosine, and N1-cyclohexylmethyl-7-(6-carbamoylpyridin-2-yl)imidazolocytosine. We further report the binding interactions of each of these analogues with the guanosine derivative 2′,3′,5′- O-tris( tert-butyldimethylsilyl)guanosine through the use of both ITC and 1H NMR titration experiments in chloroform and compare the binding to persilyated ribo-cytidine.

Production of the anti-G-quadruplex antibody BG4 for efficient genome-wide analyses: From plasmid quality control to antibody validation.

G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that can form at guanine-rich sequences of DNA and RNA in every kingdom of life. At the DNA level, G4s can form throughout genomes but they are prevalently found in promoter regions and at telomeres, and they have been attributed functions spanning from transcriptional regulation, to control of DNA replication, to maintenance of chromosome ends. Our understanding of the functions of G4s in cells has greatly improved with the development of specific anti-G4 antibodies, which allow the visualization of G4s by immunofluorescence but also the mapping of these secondary DNA structures genome wide. Whole genome identification of the location and abundance of G4s with techniques such as Chromatin Immunoprecipitation coupled with sequencing (ChIP-Seq) and Cleavage Under Target and Tagmentation (CUT&Tag) has allowed the profiling of G4 distribution across distinct cell types and deepen the understanding of G4 functions, particularly in the regulation of transcription. Crucial for these types of genome-wide studies is the availability of an anti-G4 antibody preparation with high affinity and specificity. Here, we describe a protocol for the expression and purification of the anti-DNA G4 structure antibody (BG4) first developed by the Balasubramanian group, which has been proven to selectively recognize G4 structures both in vitro and within cells, and which has great applicability in high-throughput techniques. We provide a detailed, step-by-step protocol to obtain active BG4 starting from a commercially available expression plasmid. We also describe three different approaches to validate the activity of the BG4 preparation.

G-Quadruplexes in the Viral Genome: Unlocking Targets for Therapeutic Interventions and Antiviral Strategies.

G-quadruplexes (G4s) are unique non-canonical four-stranded nucleic acid secondary structures formed by guanine-rich DNA or RNA sequences. Sequences with the potential to form quadruplex motifs (pG4s) are prevalent throughout the genomes of all organisms, spanning from prokaryotes to eukaryotes, and are enriched within regions of biological significance. In the past few years, the identification of pG4s within most of the Baltimore group viruses has attracted increasing attention due to their occurrence in regulatory regions of the genome and the subsequent implications for regulating critical stages of viral life cycles. In this context, the employment of specific G4 ligands has aided in comprehending the intricate G4-mediated regulatory mechanisms in the viral life cycle, showcasing the potential of targeting viral G4s as a novel antiviral strategy. This review offers a thorough update on the literature concerning G4s in viruses, including their identification and functional significance across most of the human-infecting viruses. Furthermore, it delves into potential therapeutic avenues targeting G4s, encompassing various G4-binding ligands, G4-interacting proteins, and oligonucleotide-based strategies. Finally, the article highlights both progress and challenges in the field, providing valuable insights into leveraging this unusual nucleic acid structure for therapeutic purposes.

Differences in Conformational Sampling and Intrinsic Electric Fields Drive Ion Binding in Telomeric and TERRA G-Quadruplexes.

The formation of G-quadruplexes (GQs) occurs in guanine-rich sequences of DNA and RNA, producing highly stable and structurally diverse noncanonical nucleic acid structures. GQs play crucial roles in regulating transcription, translation, and replication and maintaining the genome, among others; thus, changes to their structures can lead to diseases such as cancer. Previous studies using polarizable molecular dynamics simulations have shown differences in ion binding properties between telomeric and telomeric repeat-containing RNA GQs despite architectural similarities. Here, we used volume-based metadynamics and repulsive potential simulations in conjunction with polarizable force fields to quantify the impact of ion binding on the GQ dynamics and ion binding free energies. Furthermore, we describe how GQs exert electric fields on their surroundings to link dynamics with variations in the electronic structure. Our findings provide new insights into the energetic, physical, and conformational properties of GQs and expose subtle but important differences between DNA and RNA GQs with the same fold.

Identification of G4 motifs of various stem cell markers and their biophysical and biochemical characterization

Regulatory regions in the human genome, enriched in guanine-rich DNA sequences have a remarkable enrichment of G-rich sequences having a tendency to fold into G-quadruplex structures. To identify the G-quadruplex forming motifs in regulatory regions of stem cell markers, gene sequences of various stem cell markers were downloaded and analyzed to see the abundance of G-rich sequences. We observed the enrichment of G-rich sequences in stem cell markers (CD13, CD19, CD24 and CD38) which could possibly play a critical role in its regulation. We used Circular Dichroism (CD), UV-Thermal denaturation (UV-T m) and polyacrylamide gel electrophoresis (PAGE) to demonstrate the formation of a G-quadruplex by G-rich sequences present in these stem cell markers. We observed that these G-rich sequences containing minimum consecutive G3 stretch separated by loop length ranging from one to three bases long adopt G-quadruplexes with different molecularity involving two-strands, three-strand and four-strand with parallel and antiparallel conformation. Interestingly, we proposed the formation of three-stranded G-quadruplex by CD13 in 100 mM Na+, CD19 in 100 mM K+, 100 mM K+ with 40 wt% PEG 200, and CD38 in 100 mM K+ + 40 wt% PEG 200. The formation of such diverse G-quadruplex structures in the regulatory regions leaves the fair possibility of recognition by regulatory factors to modulate the gene expression. First time, this study may give insight into the structural polymorphism of G4 forming motifs in different stem cell markers to design the best suitable ligand and to target them for therapeutic development. Communicated by Ramaswamy H. Sarma

G-quadruplex resolution: From molecular mechanisms to physiological relevance

Guanine-rich DNA sequences can fold into stable four-stranded structures called G-quadruplexes or G4s. Research in the past decade demonstrated that G4 structures are widespread in the genome and prevalent in regulatory regions of actively transcribed genes. The formation of G4s has been tightly linked to important biological processes including regulation of gene expression and genome maintenance. However, they can also pose a serious threat to genome integrity especially by impeding DNA replication, and G4-associated somatic mutations have been found accumulated in the cancer genomes. Specialised DNA helicases and single stranded DNA binding proteins that can resolve G4 structures play a crucial role in preventing genome instability. The large variety of G4 unfolding proteins suggest the presence of multiple G4 resolution mechanisms in cells. Recently, there has been considerable progress in our detailed understanding of how G4s are resolved, especially during DNA replication. In this review, we first discuss the current knowledge of the genomic G4 landscapes and the impact of G4 structures on DNA replication and genome integrity. We then describe the recent progress on the mechanisms that resolve G4 structures and their physiological relevance. Finally, we discuss therapeutic opportunities to target G4 structures.

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.

Interaction of the platinum complex of tyrosine-β-cyclodextrin with G-quadruplex DNA

The telomeric quadruplex structures formed by the guanine-rich sequences of DNA have emerged as targets for small molecules designed and synthesized to stabilize the G-quadruplexes. This report presents a newly synthesized tyrosine-tethered cyclodextrin derivative and its platinum complex. Their structures are characterized using IR, NMR, and mass spectral techniques. The binding interactions of the platinum complex with CT-DNA and the kit22, myc22, and telo24 G-quadruplexes are investigated employing absorption and fluorescence spectral titrations. The binding constant or KSV values of the interaction with the G-quadruplexes are more significant than those with the duplex DNA by order of 10. It presents the compound as a G-quadruplex-selective binder. Further, the well-known G-quadruplex binding molecule Berberine is encapsulated in the Tyr- β-CD through a host: guest association. The structure of the host: guest complex is investigated employing 2D ROESY spectroscopy. In addition, the study on the binding interaction of the complex to the DNA targets is also carried out. The mode and strength of interaction of the free and the Berberine-loaded Tyr-β-CD to the duplex and the quadruplexes are reported.

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.

MOF-Based G−Quadruplex/Hemin DNAzymes for Cascade Reaction

DNA-based biomimetic enzymes have attracted extensive attention due to their unique structure and stability compared to natural enzymes. Meanwhile, the specific sequences of DNA itself also have a catalytic effect. Herein, we first designed three guanine-rich DNA sequences numbered c−Myc3c, PG4TC, and TTT to construct g−quadruplex/hemin DNAzymes. Then, the g−quadruplex/hemin DNAzymes with the best activity were selected by a comprehensive examination of activity, degradation rate, and affinity. Subsequently, the stability and reusability of UiO66−DNAzymes were investigated using UiO66 as the carrier to immobilize DNAzymes. The results showed that UiO66−DNAzymes had excellent reusability and stability. Finally, UiO66−DNAzymes were successfully used for glucose detection by cascading with glucose oxidase (GOx) with a detection limit of 0.62 μM. The constructed glucose sensor had a good specificity, which is of great significance for developing a novel, accurate, fast, and economical glucose detection sensor.

A Brief Overview of Telomeres and Telomerase in Aging and Cancer

Telomere is guanine-rich DNA sequence with a protein complex known as shelterin present at the chromosomal ends to protect it from destruction and is known to play a key role in aging. Telomerase is a telomeric DNA elongating enzyme, which comprises the central key components for telomeric DNA synthesis. The main objective of this review was to explore the structure and function of telomere and telomerase, and their intervention in aging, stem cells, and cancer cells. The induction of various telomeropathies and age-related diseases by Telomerase RNA component (TERC) impairment is well explained. Telomeropathies refer to bone marrow failures such as dyskeratosis congenita, aplastic anemia, etc. Telomere and telomerase have become the targets for age-related therapies, stem cell therapies, and cancer therapies. This article throws light on the telomerase-related therapies with natural compounds in regenerative medicine and cancer treatment. Telomerase inhibitors like nucleoside ligands and G4 ligands are explained in the context of anti-cancer drugs. The association of TERT promoter mutations with cancer is discussed. We conclude that understanding telomerase mechanisms in stem cells and cancer cells paves a way to translate stem cells, cancer cells, and age-related research into effective therapies. The dynamic features of telomerase and telomeres make their structural and biochemical studies difficult. Hence, further meticulous studies can be done on single-molecule approaches to facilitate feasible and effective studies of telomerase and telomere component functioning.

Optimization of G-Quadruplex Ligands through a SAR Study Combining Parallel Synthesis and Screening of Cationic Bis(acylhydrazones).

G-quadruplexes (G4s), secondary structures adopted by guanine-rich DNA and RNA sequences, are implicated in numerous biological processes and have been suggested as potential drug targets. Accordingly, there is an increasing interest in developing high-throughput methods that allow the generation of congeneric series of G4-targeting molecules ("ligands") and investigating their interactions with the targets. We have developed an operationally simple method of parallel synthesis to generate "ready-to-screen" libraries of cationic acylhydrazones, a motif that we have previously identified as a promising scaffold for potent, biologically active G4 ligands. Combined with well-established screening techniques, such as fluorescence melting, this method enables the rapid synthesis and screening of combinatorial libraries of potential G4 ligands. Following this protocol, we synthesized a combinatorial library of 90 bis(acylhydrazones) and screened it against five different nucleic acid structures. This way, we were able to analyze the structure-activity relationships within this series of G4 ligands, and identified three novel promising ligands whose interactions with G4-DNAs of different topologies were studied in detail by a combination of several biophysical techniques, including native mass spectrometry, and molecular modeling.