Guanine-rich Sequences Research Articles

Recent Advances in Fluorescent Probes for G-quadruplex DNAs / RNAs.

Guanine-quadruplexes (G4s) are high-level structures formed by the folding of guaninerich nucleic acid sequences. G4s play important roles in various physiological processes, such as gene transcription, replication, recombination, and maintenance of chromosomal stability. Specific and sensitive monitoring of G4s lays the foundation for further understanding the structure, content, distribution, and function of G4s in organisms, which is important for the treatment and diagnosis of diseases. Moreover, visualization of G4s will provide new ideas for developing antitumor strategies targeting G4s. The design and development of G4-specific ligands are challenging due to the subtle differences in the structure of G4s. This review focuses on the progress of research on G4 fluorescent probes and their binding mechanisms to G4s. Finally, the challenges and future prospects for better detection and targeting of G4s in different organisms are discussed. This paper provides ideas for the development of novel G4 fluorescent probes.

8-Oxoguanine DNA Glycosylase1 conceals oxidized guanine in nucleoprotein-associated RNA of respiratory syncytial virus.

Respiratory syncytial virus (RSV), along with other prominent respiratory RNA viruses such as influenza and SARS-CoV-2, significantly contributes to the global incidence of respiratory tract infections. These pathogens induce the production of reactive oxygen species (ROS), which play a crucial role in the onset and progression of respiratory diseases. However, the mechanisms by which viral RNA manages ROS-induced base oxidation remain poorly understood. Here, we reveal that 8-oxo-7,8-dihydroguanine (8-oxoGua) is not merely an incidental byproduct of ROS activity but serves as a strategic adaptation of RSV RNA to maintain genetic fidelity by hijacking the 8-oxoguanine DNA glycosylase 1 (OGG1). Through RNA immunoprecipitation and next-generation sequencing, we discovered that OGG1 binding sites are predominantly found in the RSV antigenome, especially within guanine-rich sequences. Further investigation revealed that viral ribonucleoprotein complexes specifically exploit OGG1. Importantly, inhibiting OGG1's ability to recognize 8-oxoGua significantly decreases RSV progeny production. Our results underscore the viral replication machinery's adaptation to oxidative challenges, suggesting that inhibiting OGG1's reading function could be a novel strategy for antiviral intervention.

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.

Insights into the Molecular Structure, Stability, and Biological Significance of Non-Canonical DNA Forms, with a Focus on G-Quadruplexes and i-Motifs

This article provides a comprehensive examination of non-canonical DNA structures, particularly focusing on G-quadruplexes (G4s) and i-motifs. G-quadruplexes, four-stranded structures formed by guanine-rich sequences, are stabilized by Hoogsteen hydrogen bonds and monovalent cations like potassium. These structures exhibit diverse topologies and are implicated in critical genomic regions such as telomeres and promoter regions of oncogenes, playing significant roles in gene expression regulation, genome stability, and cellular aging. I-motifs, formed by cytosine-rich sequences under acidic conditions and stabilized by hemiprotonated cytosine–cytosine (C:C+) base pairs, also contribute to gene regulation despite being less prevalent than G4s. This review highlights the factors influencing the stability and dynamics of these structures, including sequence composition, ionic conditions, and environmental pH. Molecular dynamics simulations and high-resolution structural techniques have been pivotal in advancing our understanding of their folding and unfolding mechanisms. Additionally, the article discusses the therapeutic potential of small molecules designed to selectively bind and stabilize G4s and i-motifs, with promising implications for cancer treatment. Furthermore, the structural properties of these DNA forms are explored for applications in nanotechnology and molecular devices. Despite significant progress, challenges remain in observing these structures in vivo and fully elucidating their biological functions. The review underscores the importance of continued research to uncover new insights into the genomic roles of G4s and i-motifs and their potential applications in medicine and technology. This ongoing research promises exciting developments in both basic science and applied fields, emphasizing the relevance and future prospects of these intriguing DNA structures.

Catalytic hairpin assembly-driven DNA walker to develop a label-free electrochemical aptasensor for antibiotic detection.

Anelectrochemical aptasensor was developed by utilizing a DNA walker driven by catalytic hairpin assembly (CHA) with kanamycin as the model analyte. Kanamycin bound to the aptamer, causesthe release of DNA walker, triggers the CHA reaction, leadsto the cyclic movement of the walker's long arm, and resultsin cascade amplification of thesignal. The guanine-rich sequences of the double-stranded products produced by CHA were folded to form G-quadruplex structures, with electrochemical active molecules Hemin embedded, formsG-quadruplex/Hemin complexes in situ on the electrode surface, thereby achieving sensitive, efficient, and label-free detection of kanamycin with a limit of detection (LOD) of 0.27pM (S/N = 3). Meaningfully, the aptasensor demonstrated high sensitivity and reliability in the detection of kanamycin in milk and livestock wastewater samples, suggesting that it has great potential for application in detecting antibiotics in food products and water samples from the environment.

Rapid enzyme-free detection of miRNA-21 in human ovarian cancerous cells using a fluorescent nanobiosensor designed based on hairpin DNA-templated silver nanoclusters

BackgroundCancer is known as one of the main non-communicable diseases and the leading cause of death in the new era. Early diagnosis of cancer requires the identification of special biomarkers. Currently, microRNAs (miRNAs) have attracted the attention of researchers as useful biomarkers for cancer early detection. Hence, various methods have been recently developed for detecting and monitoring miRNAs. Among all miRNAs, detection of miRNA-21 (miR-21) is important because it is abnormally overexpressed in most cancers. Here, a new biosensor based on silver nanoclusters (AgNCs) is introduced for detecting miR-21. ResultsAs a fluorescent probe, a rationally designed hairpin sequence containing a poly-cytosine motif was used to facilitate the formation of AgNCs. A guanine-rich sequence was also employed to enhance the sensing signal. It was found that in the absence of miR-21, adding a guanine-rich sequence to the detecting probe caused only a slight change in the fluorescence emission intensity of AgNCs. While in the presence of miR-21, the emission signal enhanced. A direct correlation was observed between the increase in the fluorescence of AgNCs and the concentration of miR-21. The performance of the proposed biosensor was characterized thoroughly and confirmed. The biosensor detected miR-21 in an applicable linear range from 9 pM to 1.55 nM (LOD: 2 pM). SignificanceThe designed biosensor was successfully applied for detecting miR-21 in human plasma samples and also in human normal and lung and ovarian cancer cells. This biosensing strategy can be used as a model for detecting other miRNAs. The designed nanobiosensor can measure miR-21 without using any enzymes, with fewer experimental steps, and at a low cost compared to the reported biosensors in this field.

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.

Guanine-Rich RNA Sequence Binding Factor 1 Deficiency Promotes Colorectal Cancer Progression by Regulating PI3K/AKT Signaling Pathway.

Guanine-rich RNA sequence binding factor 1 (GRSF1), part of the RNA-binding protein family, is now attracting interest due to its potential association with the progression of a variety of human cancers. The precise contribution and molecular mechanism of GRSF1 to colorectal cancer (CRC) progression, however, have yet to be clarified. Immunohistochemistry and Western Blot analysis was carried out to detect the expression of GRSF1 in CRC at both mRNA and protein levels and its subsequent effects on prognosis. A series of functional tests were performed to understand its influence on proliferation, migration, and invasion of CRC cells. The universal downregulation of GRSF1 in CRC was identified, indicating a correlation with poor prognosis. Our functional studies unveiled that the elimination of GRSF1 enhances tumour activities such as proliferation, migration, and invasion of CRC cells, while GRSF1 overexpression curtailed these abilities. Notably, we uncovered that GRSF1 insufficiency modulates the PI3K/Akt signaling pathway and Ras activation in CRC. Therefore, our data suggest GRSF1 operates as a tumor suppressor gene in CRC and may offer promise as a potential biomarker and novel therapeutic target in CRC management.

GRSF1 deficiency attenuates mitochondrial function in aging granulosa cells.

Ovarian aging results in reactive oxygen species accumulation and mitochondrial deterioration. During the aging process, GRSF1 deficiency attenuates mitochondrial function in aging granulosa cells. Ovarian aging critically influences reproductive potential, with a marked decrease in oocyte quality and quantity and an increase in oxidative stress and mitochondrial dysfunction. This study elucidates the role of guanine-rich RNA sequence binding factor 1 (GRSF1) in the aging of ovarian granulosa cells (GCs). We observed a significant reduction in GRSF1 within GCs correlating with patient age, utilizing clinical samples from IVF patients. Using an siRNA-mediated knockdown technique, we established that diminished GRSF1 expression exacerbates mitochondrial dysfunction, elevates reactive oxygen species, and impairs ATP production. Furthermore, RNA immunoprecipitation revealed GRSF1's interaction with superoxide dismutase 2 (SOD2) mRNA, a key antioxidant enzyme, suggesting a mechanism whereby GRSF1 modulates oxidative stress. Downregulation of SOD2 reversed the protective effects of GRSF1 overexpression on mitochondrial function. These insights into the role of GRSF1 in ovarian aging may guide the development of interventions to improve fertility outcomes in advanced age.

Near-infrared fluorescent probe for sensitive detection and imaging of DNA G4s in living cells

G-quadruplexs (G4s), four-stranded nucleic acid secondary structures, which formed by guanine-rich sequences play a vital role in human biological systems. Studies have shown that the formation of G4s is closely related to tumor development and apoptosis, which is considered as a new target for the development of anti-tumor drugs. Therefore, it is important to develop novel probes for G4s imaging. In this article, we engineered a near-infrared fluorescent probe (TOH) which can be activated by DNA G4s in living cells and tumor. TOH exhibits high selectivity to the structure of DNA G4s with the limit of detection for DNA G4s (Mito-0.5–2) is calculated to be 0.43 nM. Imaging studies of different cell lines revealed that the brighter fluorescence in cancer cell lines than in normal, indicating that DNA G4s maybe highly express in tumor cell lines. Simultaneously, TOH is also introduced into live tumor tissue imaging and found that the fluorescence intensity of tumor is the brightest relative to normal tissue, further validating the high expression of DNA G4s structures in tumor tissue. These features demonstrate TOH not only have the ability to image DNA G4 structures in real time, but also may have tumor diagnostic capabilities.

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.

Abstract 7118: FANCA promoteshomologous recombinationby facilitating DR-Loop formation

Abstract Homologous recombination (HR), a fundamental DNA double-strand break (DSB) repair pathway, is a universal mechanism employed by cells to accurately mend breaks in both strands of the DNA helix, ensuring genomic stability. Displacement loops, commonly referred to as D-loops, play a crucial role as intermediate structures in HR. Furthermore, recent studies indicate that the unique DNA: RNA three-stranded structure, known as R-loops, may serve as a starting point for homologous pairing and contribute significantly to enhancing HR efficiency by promoting D-loop formation. Besides, the formation of R-loops might assist in recruiting DNA repair proteins, especially those related to HR, thereby accelerating the repair of damaged sites.In this study, we find that highly purified human FANCA protein anneals synthetic single-stranded RNA (ssRNA) and ssDNA species to R-loops and binds R-loop substrates with high affinity, preferring guanine-rich sequences in vitro. In comparison, FANCA exhibits both a weaker capacity to form D-loops and a lower affinity for binding to D-loops in vitro. We then use the DART (damage at RNA transcription site) system to reveal the colocalization of FANCA with R-loops in a highly transcribed genomic locus upon DNA damage in cells. Next, we further demonstrate FANCA's participation in forming R-loops in the initial DSB repair stage and assisting in removing R-loops later. Additionally, we uncover FANCA's ability to aid in the generation of R-loops at specific DNA DSB sites within transcriptionally active regions in both Tet-DR-GFP U2OS cells and DIvA (DSB inducible via AsiSI) cells. Importantly, the R-loop formation ability of FANCA is independent of the Fanconi Anemia (FA) pathway. In conclusion, our findings indicate that FANCA, independent of the FA pathway, can enhance HR in non-transcriptionally active DNA DSB sites through D-loop formation. Furthermore, FANCA significantly elevates transcription-Coupled Homologous Recombination (TC-HR) efficiency by catalyzing the formation of DNA: RNA hybrids, generating temporary R-loops at DNA damage sites and thereby contributing to genomic stability maintenance. Citation Format: Fang Li, Haibo Yang, Boya Gao, Liang Luo, Alyan Zafar, Fenghua Yuan, Li Lan, Yanbin Zhang, Liang Luo, Alyan Zafar, Fenghua Yuan, Yanbin Zhang. FANCA promoteshomologous recombinationby facilitating DR-Loop formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7118.

Abstract 4550: Synergistic combinations of lurbinectedin with ONC212 in pancreatic cancer

Abstract Pancreatic cancer is a devastating disease with a poor five-year survival rate of approximately 12%. Given the poor prognosis of the disease, there is urgent need for novel therapeutic regimens. Lurbinectedin is chemotherapeutic compound that stalls and degrades RNA Polymerase II after binding to guanine-rich sequences in the minor groove of DNA. This stalls transcription machinery and leads to single- and double- stranded breaks in DNA and subsequent apoptosis. Our group recently described lurbinectedin’s potency as both a single agent and combinatorial agent with irinotecan and 5-fluorouracil in pancreatic cancer cell lines. We also recently discovered a synergistic interaction between lurbinectedin and ONC201/TIC10, a novel compound that induces the TRAIL apoptotic pathway, in small cell lung cancer cell lines. Synergy was accompanied by an activation of p-Chk1 and the integrated stress response. We now demonstrate a synergistic combination of lurbinectedin and ONC212, an imipridone and chemical derivative of ONC201, results in highly efficient killing of pancreatic tumor cells at sub-nanomolar concentrations of lurbinectedin and sub-micromolar concentrations of ONC212. We hypothesize that the integrated stress response underlies lurbinectedin and ONC212 synergy. We further hypothesize a combination of lurbinectedin and ONC212 will sensitize the tumor cells to CD8+ T-cell killing, which will be examined via co-culture assays. Our results are developing insights into a novel combinatorial therapeutic regimen while investigating the molecular mechanisms underlying synergism. Citation Format: Tej Tummala, Ashley S. Sevilla Uruchurtu, Nicholas R. Liguori, E. Abbas, Christopher G. Azzoli, Wafik S. El-Deiry. Synergistic combinations of lurbinectedin with ONC212 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4550.

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.

Potential protein kinase inhibitors that target G-quadruplex DNA structures in the human telomeric regions.

Telomeric regions contain Guanine-rich sequences arranged in a planar manner and connected by Hoogsteen hydrogen bonds that can fold into G-quadruplex (G4) DNA structures, and can be stabilized by monovalent metal cations. The presence of G4 DNA holds significance in cancer-related processes, especially due to their regulatory potential at transcriptional and translational levels of oncogene and tumor suppressor genes. The objective of this current research is to explore the evolving realm of FDA-approved protein kinase inhibitors, with a specific emphasis on their capacity to stabilize the G4 DNA structures formed at the human telomeric regions. This involves investigating the possibility of repurposing FDA-approved protein kinase inhibitors as a novel approach for targeting multiple cancer types. In this context, we have selected 16 telomeric G4 DNA structures as targets and 71 FDA-approved small-molecule protein kinase inhibitors as ligands. To investigate their binding affinities, molecular docking of human telomeric G4 DNA with nuclear protein kinase inhibitors and their corresponding co-crystalized ligands were performed. We found that Ponatinib and Lapatinib interact with all the selected G4 targets, the binding free energy calculations, and molecular dynamic simulations confirm their binding efficacy and stability. Thus, it is hypothesized that Ponatinib and Lapatinib may stabilize human telomeric G4 DNA in addition to their ability to inhibit BCR-ABL and the other members of the EGFR family. As a result, we also hypothesize that the stabilization of G4 DNA might represent an additional underlying mechanism contributing to their efficacy in exerting anti-cancer effects.

Stabilization of G-Quadruplex Structures of the SARS-CoV-2 Genome by TMPyP4, BRACO19, and PhenDC3.

The G-quadruplex is one of the non-canonical structures formed by nucleic acids, which can be formed by guanine-rich sequences. They became the focus of much research when they were found in several oncogene promoter regions and also in the telomeres. Later on, they were discovered in viruses as well. Various ligands have been developed in order to stabilize DNA G-quadruplexes, which were believed to have an anti-cancer or antiviral effect. We investigated three of these ligands, and whether they can also affect the stability of the G-quadruplex-forming sequences of the RNA genome of SARS-CoV-2. All three investigated oligonucleotides showed the G-quadruplex form. We characterized their stability and measured their thermodynamic parameters using the Förster resonance energy transfer method. The addition of the ligands caused an increase in the unfolding temperature, but this effect was smaller compared to that found earlier in the case of G-quadruplexes of the hepatitis B virus, which has a DNA genome.

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.