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The Neuroblastoma RAS (NRAS) oncogene homologue plays crucial roles in diverse cellular processes such as cell proliferation, survival, and differentiation. Several strategies have been developed to inhibit NRAS or its downstream effectors; however, there is no effective drug available to treat NRAS-driven cancers and thus new approaches are needed to be established. The mRNA sequence expressing NRAS containing several guanine(G)-rich regions may form quadruplex structures (G4s) and regulate NRAS translation. Therefore, targeting NRAS mRNA G4s to repress NRAS expression at translational level with ligands may be a feasible strategy against NRAS-driven cancers but it is underexplored. We reported herein a NRAS mRNA G4-targeting ligand, B3C, specifically localized in cytoplasm in HeLa cells. It effectively downregulates NRAS proteins, reactivates the DNA damage response (DDR), causes cell cycle arrest in G2/M phase, and induces apoptosis and senescence. Moreover, combination therapy with NARS mRNA G4-targeting ligands and clinical PI3K inhibitors for cancer cells inhibition treatment is unexplored, and we demonstrated that B3C combining with PI3Ki (pictilisib (GDC-0941)) showed potent antiproliferation activity against HeLa cells (IC50 = 1.03 µM (combined with 10 µM PI3Ki) and 0.42 µM (combined with 20 µM PI3Ki)) and exhibited strong synergistic effects in inhibiting cell proliferation. This study provides new insights into drug discovery against RAS-driven cancers using this conceptually new combination therapy strategy.

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Enrofloxacin (ENR) is widely used in the prevention and treatment of animal infectious diseases, so it is necessary to strengthen the residue detection of this drug in animal-derived food and water environments. In this work, for the first time, we engineered assembly a split ENR aptamer into the G-quadruplex (G4) region to form a new aptamer (G4-ENRA) that provides a more sensitive signal-reporting function while retaining target-specific recognition ability of the aptamer. This rational design effectively overcomes the issue of difficulty in identification probe development. Under the optimized conditions, a response range of 0.05-20 µM and limit of detection of 26.7 nM were obtained by directly detecting fluorescence signals, displaying a comparative advantage over the previously reported methods. Moreover, this method demonstrated satisfactory performance for the ENR detection in various real food and environmental samples, with the detection recoveries ranging from 95.87 % to 104.36 %, illustrating promising applicability prospects.

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The G-rich DNA, such as telomere, tends to form G-quadruplex (G4) structure, which slows down the replication fork progression, induces replication stress, and becomes the chromosome fragile sites. Here we described a molecular strategy that cells developed to overcome the DNA replication stress via DNA helicase regulation. The p53N236S (p53S) mutation has been found in the Werner syndrome mouse embryo fibroblast (MEFs) escaped from senescence, could be the driving force for cell escaping senescence. We revealed that the p53S could transcriptionally up-regulate DNA helicases expression, including Wrn, Blm, Timeless, Ddx, Mcm, Gins, Fanc, as well as telomere specific proteins Terf1, Pot1, through which p53S promoted the unwinding of G4 structures, and protected the cells from DNA replication stress induced by G4 stabilizer. By modified iPOND (isolation of proteins on nascent DNA) assay and telomere assay, we demonstrated that the p53S could promote the recruitment of those helicases to the DNA replication forks, facilitated the maintenance of telomere, and prevent the telomere dysfunction induced by G4 stabilizer. Interestingly, we did not observe the function of promoting G4 resolving and facilitating telomere lengthening in the cells with Li-Fraumeni Syndrome mutation-p53R172H (p53H), which suggests that this is the specific gain of function for p53S. Together our data suggest that the p53S could gain the new function of releasing the replication stress via regulating the helicase function and G4 structure, which benefits telomere lengthening. This strategy could be applied to the treatment of diseases caused by telomere replication stress.

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Mutations in the KRAS gene and overexpression of protein products of the MYC and ARF6 genes occur frequently in cancer. Here, the inseparable relationships and cooperation of the protein products of these three genes in cancer malignancy and immune evasion are discussed. mRNAs encoded by these genes share the common feature of a G-quadruplex structure, which directs them to be robustly expressed when cellular energy production is increased. These three proteins are also functionally inseparable from each other, as follows.　1) KRAS induces MYC gene expression, and may also promote eIF4A-dependent MYC and ARF6 mRNA translation, 2) MYC induces the expression of genes involved in mitochondrial biogenesis and oxidative phosphorylation, and 3) ARF6 protects mitochondria from oxidative injury. ARF6 may moreover promote cancer invasion and metastasis, and also acidosis and immune checkpoint. Therefore, the inseparable relationships and cooperation of KRAS, MYC, and ARF6 appear to result in the activation of mitochondria and the driving of ARF6-based malignancy and immune evasion. Such adverse associations are frequent in pancreatic cancer, and appear to be further enhanced by TP53 mutations. Video Abstract.

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Identification of a new G-quadruplex ligand having anti-telomerase activity would be a promising strategy for cancer therapy. The screened compound from ZINC database using docking studies was experimentally verified for its binding with three different telomeric G-quadruplex DNA sequences and anti-telomerase activity in A549 cells. Identified compound is an intrinsic fluorescent molecule, permeable to live cells and has a higher affinity to 22AG out of three different telomeric G-quadruplex DNA. It showed cytotoxicity and a significant reduction of telomerase activity in human A549 cells at a very low dose. So, this compound has a good anti-cancer effect.

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The structural polymorphism of G-quadruplex nucleic acids is an important factor in their recognition by proteins and small-molecule ligands. However, it is not clear why the binding of several ligands alters G-quadruplex topology. We addressed this question by following the (un)folding and binding of the human telomeric fragment 5'-(GGGTTA)3GGGT-3' (22GT) by calorimetry (DSC, ITC) and spectroscopy (CD). A thermodynamic analysis of the obtained data led to a detailed description of the topological phase space of stability (phase diagram) of 22GT and shows how it changes in the presence of a specific bisquinolinium ligand (360A). Various 1:1 and 2:1 ligand-quadruplex complexes were observed. With increasing temperature, the 1:1 complexes transformed into 2:1 complexes, which is attributed to the preferential binding of the ligand to the folding intermediates. Overall, the dissection of the thermodynamic parameters in combination with molecular modelling clarified the driving forces of the topological quadruplex transformations in a wide range of ligand concentrations and temperatures.

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Damaging the structure of the G-quadruplex (G4) to prevent the formation of the G4/hemin complex is presently the only available method to inhibit the activity of the peroxidase-mimic DNAzyme. In this study, a unique intramolecular inhibitory effect of the adjacent base-pair (InE(N:N)), by installing a rationally adjacent base-pair of the G4 core sequence, is proposed for the inhibition of the DNAzyme activity, which eliminates the need to damage the entire G4 structure. Various base pairs show different abilities to inhibit DNAzyme activity. The adjacent adenine: thymine pair possesses the best inhibitory efficiency (17 times). Through detailed investigations of the InE(N:N), it was revealed that the adjacent adenine: thymine pair downregulated the formation of compound I in the catalytic process, thus inhibiting the G4 DNAzyme activity. The mechanism of inhibition indicated that the carbonyl group on the hexatomic ring of the complementary base played an important role. To further reflect the advantages of the proposed strategy, two InE(N:N)-based biosensors were developed for DNA analysis and Uracil-DNA glycosylase (UDG) detection. Compared with existing DNAzyme-based methods, the application of InE(N: N) facilitates the real-time assay and simplifies the design difficulty. Therefore, InE(N:N) provides new insights into the regulation of the DNAzyme activity and offers an efficient approach for the future application of DNAzyme.

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RNA structure plays key roles in plant growth, development, and adaptation. One of the complex RNA structures is the RNA G-quadruplex (RG4) where guanine-rich sequences are folded into two or more layers of G-quartets. Previous computational predictions of RG4 revealed that it is widespread across the whole transcriptomes in many plant species, raising the hypothesis that RG4 is likely to be an important regulatory motif in plants. Recently, with the advances in both high-throughput sequencing and cell imaging technologies, RG4 can be detected in living cells as well as at the genome-wide scale. Here, we provide a comprehensive review of recent developments in new methods for detecting RG4 in plants. We also summarize the new functions of RG4 in regulating plant growth and development. We then discuss the possible role of RG4 in adapting to environmental conditions along with evolutionary perspectives.

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Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies.

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BACKGROUND: Cell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations. RESULTS: We aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3'-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments. CONCLUSIONS: We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

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G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.

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Zika virus (ZIKV), a mosquito-transmitted Flavivirus, emerged in the last decade causing serious diseases and affecting human health globally. Currently, no licensed vaccines or antivirals are available to combat ZIKV, although several vaccine candidates are in the pipeline. In recent years, the presence of non-canonical G-quadruplex (GQ) secondary structures in viral genomes has ignited significant attention as potential targets for antiviral strategy. In this study, we identified several novel conserved potential GQ structures by analyzing published ZIKV genome sequences using an in-house algorithm. Biophysical and biochemical analysis of the RNA sequences containing these potential GQ sequences suggested the existence of such structures in the ZIKV genomes. Studies with known GQ structure-binding and -stabilizing ligands such as Braco-19 and TMPyP4 provided support for this contention. The presence of these ligands in cell culture media led to significant inhibition of infectious ZIKV yield, as well as reduced viral genome replication and viral protein production. Overall, our results, for the first time, show that ZIKV replication can be inhibited by GQ structure-binding and -stabilizing compounds and suggest a new strategy against ZIKV infection mitigation and control.

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RNA G-quadruplex (rG4) structure and its association with rG4-binding proteins/peptides are important for its function. However, there is very limited study that investigates what factors are involved in rG4 that drive the rG4-protein/peptide interaction. Here we study and uncover the effect of RNA sequence context and stereochemistry on G-quadruplex-peptide interaction. Using rG4-binding RHAU53 peptide as an example, we report that the number of G-quartet, thermostability, overhanging nucleotides, and RNA base chirality have an impact on rG4-RHAU53 binding. Notably, our data also demonstrate that RHAU53 preferentially binds to 5' G-quartet over 3' G-quartet, and showcase that RHAU53 interacts with unnatural L-rG4 for the first time. Our findings reported here offer unique insights to the potential development of targeting tools that recognize rG4 structure and rG4-binding peptide/protein.

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BACKGROUND: Guanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA G-quadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA G-quadruplex formation in living eukaryotic cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure. RESULTS: Using rG4-seq, we profile the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We find a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determine that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analyses show that RNA G-quadruplex folding is able to regulate translation and modulate plant growth. CONCLUSIONS: Our study reveals the existence of RNA G-quadruplex in vivo and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

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SNAIL1 is a key regulator of epithelial-mesenchymal transition (EMT) and its expression is associated with tumor progression and poor clinical prognosis of cancer patients. Compared to the studies of SNAIL1 stability and its transcriptional regulation, very limited knowledge is available regarding effective approaches to directly target SNAIL1. In this study, we revealed the potential regulation of SNAIL1 gene expression by G-quadruplex structures in its promoter. We first revealed that the negative strand of the SNAIL1 promoter contained a multi-G-tract region with high potential of forming G-quadruplex structures. In circular dichroism studies, the oligonucleotide based on this region showed characteristic molar ellipticity at specific wavelengths of G-quadruplex structures. We also utilized native polyacrylamide gel electrophoresis, gel-shift assays, immunofluorescent staining, dimethyl sulfate footprinting and chromatin immunoprecipitation studies to verify the G-quadruplex structures formed by the oligonucleotide. In reporter assays, disruption of G-quadruplex potential increased SNAIL1 promoter-mediated transcription, suggesting that G-quadruplexes played a negative role in SNAIL1 expression. In a DNA synthesis study, we detected G-quadruplex-mediated retardation in the SNAIL1 promoter replication. Consistently, we discovered that the G-quadruplex region of the SNAIL1 promoter is highly enriched for mutations, implicating the clinical relevance of G-quadruplexes to the altered SNAIL1 expression in cancer cells.

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The interaction between thrombin binding aptamer (TBA) and thrombin (TB) was studied by resonance Rayleigh scattering (RRS). In neutral medium, TBA is present in a balanced form between a G-quadruplex structure and a random coil structure, and the TBA can be induced by metal ions to form a G-quadruplex structure. Upon addition of thrombin, the G-quadruplex selectively bound to TB, which resulted in enhanced resonance Rayleigh scattering. The scattering intensities increased proportionally with the concentration of TB from 10 to 50 nM. The method had very high sensitivity and good selectivity, and the detection limit (3δ/s) was 1 nM. In this work, the spectral characteristics of RRS, the optimum conditions of the reaction, and influencing factors for the RRS intensities were investigated. Furthermore, the structure of the TBA-TB complex and the sensing mechanism were explored. The TB sensor was applied to a diluted human serum sample with satisfactory results, indicating the potential of this method to be applied to biological samples. A selective and simple RRS sensor for the detection of trace amounts of TB is proposed based on conformational change of TBA.

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In this study, a simple, label-free, and enzyme-free colorimetric biosensor has been developed for amplified detection of let-7a microRNA (miRNA) on the basis of dual signal amplification strategy. The sensing system mainly consists of four unlabeled hairpin probes termed H1, H2, H3, and H4. Upon sensing of the target miRNA, hairpin H1 is opened. Then hairpin H2 hybridizes with H1 forming H1-H2 duplex and frees the target miRNA that can be recycled to trigger another reaction cycle. In addition, the newly formed H1-H2 duplex hybridizes with hairpin H3, and this triggers the autonomous cross-opening of the two hairpins H3 and H4 through hybridization chain reaction. During this process, numerous split G-quadruplex structures are generated and further associate with cofactor hemin to form massive peroxidase-mimicking DNAzymes. The resulting DNAzymes catalyze the H2O2-mediated oxidation of colorless 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(2-)) to the green-colored ABTS(•-), inducing a remarkably amplified colorimetric signal. This newly developed sensing system exhibits high sensitivity toward miRNA with a detection limit of 7.4fM and a large dynamic range of 6 orders of magnitude from 10fM to 10nM. Furthermore, it exhibits a good performance to discriminate single-base difference among the miRNA family members and holds a great potential for early diagnosis in gene-related diseases.

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Previous studies have shown that promoter regions of many proto-oncogenes can fold into G-quadruplexes, which are potentially involved in the regulation of genes. Bioinformatics analysis suggested that there was a G-rich sequence within -48 to -26 region of the human MET promoter (named Pu23WT). In this study, we proved that Pu23WT adopted an intramolecular parallel G-quadruplex structure under physiological conditions in vitro, and the cationic porphyrin TMPyP4 enhanced the stability of the Pu23WT G-quadruplex. To better understand the functions of Pu23WT in the MET expression, we performed a series of analysis on several cancer cells. Experimental data revealed that TMPyP4 treatment attenuated the expression of MET in HepG2, BGC823, and U87MG cells, resulting in the cellular proliferation inhibition, G1 phase cell cycle arrest and cell migration retardation. ChIP assay results indicated that TMPyP4 probably prohibited the Pu23WT G-quadruplex from binding to the activator Sp1, which could be one of the mechanisms that led to the transcription inhibition of MET gene. It is the first study on the G-quadruplex structure in the human MET promoter and its functions in cancer cells. We believe that this structure is a potential target for anticancer treatment.

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Thrombin binding G-quadruplex oligonucleotide containing two TT-dimer fragments and a gold attachment (ODN G1-G) was designed and synthesized with the aim of understanding the TT-dimer effect in G-quadruplex formation. Our results showed that TT-dimer mutation induced by UV light inhibits the formation of and even destroys the G-quadruplex structure, as confirmed by UV, CD and melting temperature measurements. The structural change resulting from TT-dimer formation with DNA was additionally probed and was found to be accompanied by significant gold aggregation that was observed in the form of a signal change from red to blue.

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