Telomeres play an important role in cellular aging and cancer. Human telomeric DNA and RNA G-rich sequences are capable of forming a four-stranded structure, known as the G-quadruplex. Such a ...structure might be important for telomere biology and a good target for drug design. This minireview describes the structural diversity or conservation of DNA and RNA human telomeric G-quadruplexes, discusses structural views on targeting these G-quadruplexes and presents some future challenges for structural studies.
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.
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.
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.
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.
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.
An efficient inhibitory effect of adjacent base-pair was found that the inhibition of peroxidase-mimic DNAzyme activity does not require to damage G-quadruplex structure. Display omitted
•The intramolecular inhibitory effect of adjacent base-pair (InE@N:N) to inhibit peroxidase-mimic DNAzyme activity was found.•InE@N:N does not necessarily damage the G-quadruplex structure.•Adjacent adenine:thymine pair possesses the best inhibition efficiency.•The InE@N:N-based biosensing strategy is real-time and simple.
•A simple, label-free and turn-on fluorescence method was presented.•The NMM/G-quadruplex structure is used as a label-free signal reporter.•The sensitivity of this method was very high and the ...detection limit was 12.5 nM.•This method was applied to detect melamine in raw milk and milk powder.
Melamine has a high nitrogen content thus is often illegally added to milk powder to phonily boost the protein percentage composition. Milk products containing melamine are harmful to babies and children. In the present work, a simple, label-free and turn-on fluorescence method to detect melamine with a high sensitivity and selectivity has been developed. The N-methylmesoporphyrin IX (NMM)/G-quadruplex structure is used as a signal reporter in this method to realize label-free detection of melamine. The detection limit of this method is 12.5 nM, which is much less than the maximum level permitted for infant formulas. The linear range is 0.05–1 μM (R2 = 0.98). The selectivity of this method is also very satisfactory. Furthermore, this method can be successfully applied for detection melamine in raw milk and milk powder.
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•A label-free and simple split aptamer-based fluorescent sensor was proposed for cocaine detection.•The aptasensor used N-methyl mesoporphyrin IX (NMM)/G-quadruplex system as the ...fluorescent indicator.•This aptasensor is cost-effective, simple, and does not require chemical modification with fluorescence group.•The biosensor exhibited a useful linear concentration range from 50 pM to 25 nM and a limit of detection of 16 pM.•The applied potential of the aptasensor was demonstrated by cocaine detection in serum samples.
Cocaine is an extensively used illicit drug worldwide. So, the development of a sensing method for cocaine detection is necessary for examination and lawful action against drug trafficking and abuse. A label-free and simple split aptamer-based fluorescent aptasensor was proposed for cocaine detection using the N-methyl mesoporphyrin IX (NMM)/G-quadruplex system as a fluorescent indicator. The working principle of the developed aptasensor is an increase in the emission intensity of fluorescence of the NMM in the attendance of cocaine due to the formation of split G-quadruplex structure as a suitable platform for attachment of NMM. Under optimized conditions, the designed fluorescent aptasensor exhibited a useful linear concentration range from 50 pM to 25 nM and a limit of detection (LOD) of 16 pM. The applied potential of the designed fluorescent aptasensor was demonstrated by cocaine detection in spiked human serum samples with satisfying recoveries.
•An enrofloxacin detection method based on G-quadruplex structure-switching aptamer have been constructed for the first time.•The novel G-quadruplex structure-switching aptamer retains high affinity ...and strong specificity for the target.•The detection method has the advantage of sensitivity and a broad detection range.•This proposed sensor was successfully achieved the quantitative analysis of enrofloxacin in various food and environmental samples.
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.
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) (ABTS2−) 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.
•A label-free and enzyme-free colorimetric miRNA biosensor has been developed.•Catalyzed hairpin assembly and hybridization chain reaction are perfectly combined.•A low detection limit of 7.4 fM toward target miRNA can be achieved.•The sensing system exhibits a good performance to discriminate single-base difference among the miRNA family members.
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.
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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G-quadruplex structures are non-canonical secondary structures present in nucleic acid. In this manuscript, we targeted the Zika GQ structures with GQ-binding ligands like Braco-19 and TMPyP4 to inhibit ZIKV genome replication and transcription. Overall, 100 μM Braco-19 treatment produced >80-fold (96 hpi) reduction in viral titer production in cell culture, having a potential anti-viral effect.
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