G-quadruplex Formation Research Articles

Cationic porphyrin TMPyP4 redox behaviour and interaction with nucleic acids: Towards a new methodology for screening porphyrin-based anticancer drugs

The cationic meso-substituted porphyrin 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin (TMPyP4) is considered an excellent model compound for the development of new porphyrin-based anticancer drugs able to inhibit the telomerase in cancer cells. Drug development testing methods are nowadays laborious and time-consuming; hence, the development of rapid, sensitive, selective, low-cost techniques of testing the effectiveness of drugs acting on specific DNA sequences is essential. TMPyP4 oxidation behavior and its interaction with different nucleic acid sequences were electrochemically investigated, aiming to develop an easy, label free and low cost electrochemical methodology of fast screening porphyrin-based anticancer drugs, by differentiating their affinity to different nucleic acid structures.TMPyP4 undergoes an irreversible, pH dependent, two-step oxidation at glassy carbon electrode, each step occurring with the loss of one electron and one proton from the nitrogen atoms of the porphine central ring. DPV was able to discriminate between TMPyP4 binding to different nucleic acid sequences, in agreement with models existent in the literature: (i) TMPyP4 interaction with DNA double-helix is fast and occurs by intercalation, preferentially at the G-C base pairs, but does not cause oxidative DNA damage, (ii) TMPyP4 interaction with polyadenylic acid single-helices formed at neutral pH occurs by pseudo-intercalation, and with single- and double-helices formed at mild acidic pH occurs by side-binding, and (iii) TMPyP4 interaction with polyguanylic acid promotes the formation of G-quadruplexes. The voltammetric results represent a proof of concept, confirming the ability of the proposed electrochemical methodology to screen porphyrin-based anticancer drugs in the process of drug development, allowing the drug easy classification in terms of intercalation and selectivity for different nucleic acid structures.

Advanced Binary Guanosine and Guanosine 5'-Monophosphate Cell-Laden Hydrogels for Soft Tissue Reconstruction by 3D Bioprinting.

Soft tissue defects or pathologies frequently necessitate the use of biomaterials that provide the volume required for subsequent vascularization and tissue formation as autrografts are not always a feasible alternative. Supramolecular hydrogels represent promising candidates because of their 3D structure, which resembles the native extracellular matrix, and their capacity to entrap and sustain living cells. Guanosine-based hydrogels have emerged as prime candidates in recent years since the nucleoside self-assembles into well-ordered structures like G-quadruplexes by coordinating K+ ions and π-π stacking, ultimately forming an extensive nanofibrillar network. However, such compositions were frequently inappropriate for 3D printing due to material spreading and low shape stability over time. Thus, the present work aimed to develop a binary cell-laden hydrogel capable of ensuring cell survival while providing enough stability to ensure scaffold biointegration during soft tissue reconstruction. For that purpose, a binary hydrogel made of guanosine and guanosine 5'-monophosphate was optimized, rat mesenchymal stem cells were entrapped, and the composition was bioprinted. To further increase stability, the printed structure was coated with hyperbranched polyethylenimine. Scanning electron microscopic studies demonstrated an extensive nanofibrillar network, indicating excellent G-quadruplex formation, and rheological analysis confirmed good printing and thixotropic qualities. Additionally, diffusion tests using fluorescein isothiocyanate labeled-dextran (70, 500, and 2000 kDa) showed that nutrients of various molecular weights may diffuse through the hydrogel scaffold. Finally, cells were evenly distributed throughout the printed scaffold, cell survival was 85% after 21 days, and lipid droplet formation was observed after 7 days under adipogenic conditions, indicating successful differentiation and proper cell functioning. To conclude, such hydrogels may enable the 3D bioprinting of customized scaffolds perfectly matching the respective soft tissue defect, thereby potentially improving the outcome of the tissue reconstruction intervention.

G-Quadruplexes Formation by the C9orf72 Nucleotide Repeat Expansion d(GGGGCC)n and Conformation Regulation by Fangchinoline.

The G-quadruplex (GQ)-forming hexanucleotide repeat expansion (HRE) in the C9orf72 (C9) gene has been found to be the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (collectively, C9ALS/FTD), implying the great significance of modulating C9-HRE GQ structures in C9ALS/FTD therapeutic treatment strategies. In this study, we investigated the GQ structures formed by varied lengths of C9-HRE DNA sequences d(GGGGCC)4 (C9-24mer) and d(GGGGCC)8 (C9-48mer), and found that the C9-24mer forms anti-parallel GQ (AP-GQ) in the presence of potassium ions, while the long C9-48mer bearing eight guanine tracts forms unstacked tandem GQ consisting of two C9-24mer unimolecular AP-GQs. Moreover, the natural small molecule Fangchinoline was screened out in order to be able to stabilize and alter the C9-HRE DNA to parallel GQ topology. Further study of the interaction of Fangchinoline with the C9-HRE RNA GQ unit r(GGGGCC)4 (C9-RNA) revealed that it can also recognize and improve the thermal stability of C9-HRE RNA GQ. Finally, use of AutoDock simulation results indicated that Fangchinoline binds to the groove regions of the parallel C9-HRE GQs. These findings pave the way for further studies of GQ structures formed by pathologically related long C9-HRE sequences, and also provide a natural small-molecule ligand that modulates the structure and stability of C9-HRE GQ, both in DNA and RNA levels. Altogether, this work may contribute to therapeutic approaches of C9ALS/FTD which take the upstream C9-HRE DNA region, as well as the toxic C9-HRE RNA, as targets.

Dual targets-induced specific hemin/G-quadruplex assemblies for label-free electrochemical detection capable of distinguishing Salmonella and its common serotype in food samples

Efficient detection of pathogenic bacteria is paramount for ensuring food safety and safeguarding public health. Herein, we developed a label-free and signal-on dual-target recognition electrochemical DNA sensing platform based on the conformational formation of split G-quadruplex. This platform focused on achieving sensitive and low-cost detection of Salmonella and its most human-infecting S. typhimurium serotype. In simple terms, the dual-target recognition probe (DTR-6P) was ingeniously designed for the loop sequence on the loop-mediated isothermal amplification (LAMP) amplicons. It could recognize two different genes and release their corresponding G-rich sequences. The exfoliated G-rich sequences could be captured by the capture probes on the electrode, and then the bimolecular G-quadruplex or the tetramolecular G-quadruplex would be formed to capture hemin, thereby enabling dual-signal reporting. The minimum detection amount of target genes can be as low as 2 copies/μL. Encouragingly, the real food samples contaminated by Salmonella and the S. typhimurium serotype can be readily identified. The sensing platform with ingenious design paves a new way for label-free, multi-target simultaneous detection, whose advantage of rapidity, sensitivity, cost-effectiveness, and specificity also lay a solid foundation for practical applications.

A cyanine dye probe for K+ detection based on DNA construction of G-quadruplex.

Potassium ion (K+) plays an important role in the maintenance of cellular biological process for human health. Thus, the detection of K+ is very important. Here, based on the interaction between thiamonomethinecyanine dye and G-quadruplex formation sequence (PW17), K+ detection spectrum was characterized by UV-Vis spectrometry. The single-stranded sequence of PW17 can fold into G-quadruplex in the presence of K+. PW17 can induce a dimer-to-monomer transition of the absorption spectrum of cyanine dyes. This method shows high specificity against some other alkali cations, even at high concentrations of Na+. Further, this detection strategy can realize the detection of K+ in tap water.

Mapping and characterization of G-quadruplexes in monkeypox genomes.

Monkeypox virus (MPXV) is a double-stranded DNA virus from the family Poxviridae, which is endemic in West and Central Africa. Various human outbreaks occurred in the 1980s, resulting from a cessation of smallpox vaccination. Recently, MPXV cases have reemerged in non-endemic nations, and the 2022 outbreak has been declared a public health emergency. Treatment optionsare limited, and many countries lack the infrastructure to provide symptomatic treatments. The development of cost-effective antivirals could ease severe health outcomes. G-quadruplexes have been a target of interest in treating viral infections with different chemicals. In the present work, a genomic-scale mapping of different MPXV isolates highlighted two conserved putative quadruplex-forming sequences MPXV-exclusive in 590 isolates. Subsequently, we assessed the G-quadruplex formation using circular dichroismspectroscopy and solution small-angle X-ray scattering. Furthermore, biochemical assays indicated the ability of MPXV quadruplexes to be recognized by two specific G4-binding partners-Thioflavin T and DHX36. Additionally, our work also suggests that a quadruplex binding small-molecule with previously reported antiviral activity, TMPyP4, interacts with MPXV G-quadruplexes with nanomolar affinity in the presence and absence of DHX36. Finally, cell biology experiments suggests that TMPyP4 treatment substantially reduced gene expression of MPXV proteins. In summary, our work provides insights into the G-quadruplexes from the MPXV genome that can be further exploited to develop therapeutics.

Dynamic Multicomponent Reactions-Directed Self-Assembled G-quadruplex Inherent Antibacterial Hydrogel.

Nowadays, inherent antibacterial hydrogels have gained significant attention due to their utilization against infectious bacteria. Herein, we focus on the development of an injectable, self-healable, dynamic, and G-quadruplex hydrogel with inherent antibacterial activity. The dynamic self-assembled hydrogel is constructed upon multicomponent reactions (MCR) among guanosine, 2-formylphenylboronic acid, and amino acid/peptides in the presence of potassium ions. The role of amino acid/peptides in the formation of the G-quadruplex hydrogel is studied in detail. The G-quadruplex structure is formed via the π-π stacking of G-quartets. The formation of G-quadruplex is investigated by thioflavin T binding assay, CD spectroscopy, and PXRD. The formation of the dynamic imino-boronate bond in the hydrogels is well characterized by temperature-dependent 11B NMR (VT-NMR) and FT-IR spectroscopy. Furthermore, HR-TEM images and rheological experiments reveal the fibrillar networks and viscoelastic property of the hydrogels. The presence of the dynamic imino-boronate ester bonds makes the hydrogel injectable and self-healable in nature. These dynamic G-quadruplex hydrogels show potential antibacterial activity against a series of Gram-positive and Gram-negative bacteria. The hydrogels have been used for the entrapment and sustained release of an anticancer drug doxorubicin over 48 h at different pHs (4.8, 7.4, and 8.5) and temperature without the influence of any external stimuli. Such injectable and self-healable hydrogels could be used in various applications in the field of biomedical science.

The fluorescence regulation of a tri-functional oligonucleotide probe HEX-OND in detecting Pb(II), cysteine, and K(I) based on two G-quadruplex forms.

A novel tri-functional probe HEX-OND was developed for detecting Pb(II), cysteine (Cys), and K(I) by fluorescence quenching, recovery, and amplification strategies respectively, based on Pb(II)-induced chair-type G-quadruplex (CGQ) and K(I)-induced parallel G-quadruplex (PGQ). The thermodynamic mechanism was illustrated as that HEX-OND transformed into CGQ by associating equimolar Pb(II) (K1 = 1.10 ± 0.25 × 106 L/mol), forcing (G)2 spontaneously approaching and static-quenching HEX (5'-hexachlorofluorescein phosphoramidite) in the photo-induced electron transfer (PET) way by the van der Waals force and hydrogen bond (K2 = 5.14 ± 1.65 × 107 L/mol); the additional Cys recovered fluorescence in the molecular ratio of 2:1 via Pb(II)-precipitation induced CGQ destruction (K3 = 3.03 ± 0.77 × 109 L/mol); the equimolar K(I) induced HEX-OND transforming into PGQ (K4 = 3.53 ± 0.30 × 104 L/mol) and specifically associating with the equimolar N-methyl mesoporphyrin IX (NMM) by hydrophobic force (K5 = 3.48 ± 1.08 × 105 L/mol), leading to the fluorescence enhancement. Moreover, the practicability results showed that the detection limits reached a nanomolar level for Pb(II) and Cys and micromolar for K(I), with mere disturbances for 6, 10, and 5 kinds of other substances, respectively; no significant deviations of the real sample detection results were found between the well-understood methods with ours in detecting Pb(II) and Cys, and K(I) could be recognized and quantified even in the presence of Na(I) with 5000 and 600 fold respectively. The results demonstrated the triple-function, sensitivity, selectivity, and tremendous application feasibility of the current probe in sensing Pb(II), Cys, and K(I).

Colorimetric analysis of sulfate-reducing bacterial DNA based on catalytic hemin/G-quadruplex loaded rigid DNA triangle

Here a colorimetric sensing platform for sulfate-reducing bacterial DNA analysis was described by using a catalytic hemin/G-quadruplex loaded rigid DNA triangle assembly. This two-dimensional rigid DNA triangle structure was assembled by target bacterial DNA and three hairpin beacon probes. As three tips of hairpin probes carry specific recognition sites for G-quadruplex formation, target bacterial DNA trigger the opening of hairpin probes, not only forming a steady rigid DNA triangle but also activating the hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme. The resultant hemin/G-quadruplex loaded rigid DNA triangle assembly catalyzes a colorimetric reaction with the signal intensity correlated to target concentrations, achieving target bacterial DNA detection with a detection limit as low as 23.99 fM. We further analyzed actual bacteria with this system, by acquiring bacterial DNA including bacteria-specific sequences with polymerase chain reaction (PCR), with the detection limit down to 1 cfu/mL. The system shows good selectivity for the target and resistance to interference, highly stable performance for serum samples application, showing great potential for application in the fields of genetic analysis, environmental monitoring, and medical testing.

G-QINDER Tool: Bioinformatically Predicted Formation of Different Four-Stranded DNA Motifs from (GT)n and (GA)n Repeats

The recently introduced semi-orthogonal system of nucleic acid imaging offers a greatly improved method of identifying DNA sequences that are capable of adopting noncanonical structures. This paper uses our newly developed G-QINDER tool to identify specific repeat sequences that adopt unique structural motifs in DNA: TG and AG repeats. The structures were found to adopt a left-handed G-quadruplex form under extreme crowding conditions and a unique tetrahelical motif under certain other conditions. The tetrahelical structure likely consists of stacked AGAG-tetrads but, unlike G-quadruplexes, their stability does not appear to be dependent on the type of monovalent cation present. The occurrence of TG and AG repeats in genomes is not rare, and they are also found frequently in the regulatory regions of nucleic acids, so it is reasonable to assume that putative structural motifs, like other noncanonical forms, could play an important regulatory role in cells. This hypothesis is supported by the structural stability of the AGAG motif; its unfolding can occur even at physiological temperatures since the melting temperature is primarily dependent on the number of AG repeats in the sequence.

Stabilization of the Quadruplex-Forming G-Rich Sequences in the Rhinovirus Genome Inhibits Uncoating-Role of Na+ and K.

Rhinoviruses (RVs) are the major cause of common cold, a respiratory disease that generally takes a mild course. However, occasionally, RV infection can lead to serious complications in patients debilitated by other ailments, e.g., asthma. Colds are a huge socioeconomic burden as neither vaccines nor other treatments are available. The many existing drug candidates either stabilize the capsid or inhibit the viral RNA polymerase, the viral proteinases, or the functions of other non-structural viral proteins; however, none has been approved by the FDA. Focusing on the genomic RNA as a possible target for antivirals, we asked whether stabilizing RNA secondary structures might inhibit the viral replication cycle. These secondary structures include G-quadruplexes (GQs), which are guanine-rich sequence stretches forming planar guanine tetrads via Hoogsteen base pairing with two or more of them stacking on top of each other; a number of small molecular drug candidates increase the energy required for their unfolding. The propensity of G-quadruplex formation can be predicted with bioinformatics tools and is expressed as a GQ score. Synthetic RNA oligonucleotides derived from the RV-A2 genome with sequences corresponding to the highest and lowest GQ scores indeed exhibited characteristics of GQs. In vivo, the GQ-stabilizing compounds, pyridostatin and PhenDC3, interfered with viral uncoating in Na+ but not in K+-containing phosphate buffers. The thermostability studies and ultrastructural imaging of protein-free viral RNA cores suggest that Na+ keeps the encapsulated genome more open, allowing PDS and PhenDC3 to diffuse into the quasi-crystalline RNA and promote the formation and/or stabilization of GQs; the resulting conformational changes impair RNA unraveling and release from the virion. Preliminary reports have been published.

Abstract 1436: T-oligo's mechanism of action in melanoma cells

Abstract Telomeres have become an attractive target for anticancer therapeutics and in 2022, an estimated 99,780 new cases and 7,650 people are expected to die of melanoma. T-oligo, a hexanucleotide tandem repeat oligonucleotide that is homologous to the 3′ overhang of telomeres has been shown to have potent anticancer effects in-vitro by inhibiting the growth of cancer cells. There are three mechanisms of action proposed for T-oligo, which are shelterin dissociation model, exposed telomere mimicry model, and G-quadruplex formation. Previous studies utilizing NMR spectroscopy and immunofluorescence assays have shown that T-oligo forms G-quadruplexes in the nucleus of the melanoma cells. We hypothesize that formation of G-quadruplexes can be detected with the help of FRET analysis and T-oligo could be delivered using lipid nanoparticles. Immunofluorescence assays and an acceptor photobleaching method were used to detect FRET and confirm the formation of G-quadruplexes in the nucleus of melanoma cells. Melanoma cells were plated and grown until 70% confluent in 8-well glass chamber slides and were treated with single-labeled FITC-T-oligo (11-mer), and Cy3-T-oligo (11-mer), and double labeled T-oligo 11-mer (T-11) and 22-mer (T-22) labeled with FITC on the 5′ end and Cy3 on the 3′ end. Slides were mounted and cells were observed under a fluorescent microscope. For FRET detection, we used the acceptor photobleaching method in which the acceptor (Cy3) was completely photobleached and any corresponding increase in donor’s intensity was detected and quantified. Any significant increase in donor intensity was considered to be due to FRET. We also performed some preliminary experiments with lipid nanoparticles to study whether these lipid nanoparticles are a viable delivery system for T-oligo. We encapsulated T-oligo in lipid nanoparticles using a microfluidic mixer and studied the encapsulation efficiency, uptake, and efficacy of encapsulated T-11 and T-22 on MM-AN melanoma cells. We found a significant increase of 40% - 150% in donor intensity after acceptor photobleaching in multiple melanoma cell lines. This may be due to G-quadruplex formation causing FRET thus indicating that T-oligo may be utilizing this mechanism of action for its anticancer effects. Preliminary results indicate that lipid nanoparticles encapsulated T-oligo with an efficiency of 72%. Uptake experiments showed that encapsulated T-11 and T-22 were taken up by the cells after an incubation time of 6hrs and the encapsulated T-22 also inhibited cell proliferation by 92%. In conclusion, three melanoma cell lines showed a significant increase in donor intensity after acceptor photobleaching upon treatment with T-22 and T-11 and lipid nanoparticles might be an effective delivery system for T-oligo. Citation Format: Archit Agnihotri, Taylor Rager, Kelly Fan, Hyatt Alaraj, Lawrence Miller, Neelu Puri. T-oligo's mechanism of action in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1436.

Supramolecular G-quadruplex hydrogels: Bridging fabrication to biomedical application

G-quadruplex hydrogel is a class of self-assembled supramolecular hydrogel formed by guanine derivatives. As a biomimetic hydrogel, G-quadruplex hydrogels demonstrate wide biomedical applications, such as drug delivery, tissue engineering, and biosensing. The advantages of using G-quadruplex hydrogels include adequate biocompatibility and biodegradability, tunable multifunctionality, and cost-effective and large-scalable fabrication process. In this review, we focus on recent progress in the fabrication and characterization of G-quadruplex hydrogels to help readers understand the principles of G-quadruplex hydrogel formation. Meanwhile, the applications of G-quadruplex hydrogels in the biomedical area are discussed, aiming to pave the way for downward clinical or industry translation. The development of G-quadruplex hydrogel is still in its infancy. We hope this review will boost the development of this area and that more applications of G-quadruplex hydrogel will be developed.

Interaction of dpyatriz and Cu/Zn-dpyatriz complexes with human telomere DNA: The role of G-quadruplex formation and its effect on antitumor and antitelomerase activity

1,3,5-Triazine derivative of dipyridyl amine (dpyatriz) and the Cu(II), Zn(II) complexes have been prepared, characterized by CHN, IR, NMR and mass measurements and interacted with HTelo8 and HTelo20, the two types of human telomere repeat DNA (TTAGGG)n; n = 8, 20. The telomere DNAs have been treated with all three compounds (dpyatriz, Cu-dpyatriz and Zn-dpyatriz) under parallel/antiparallel and random coiled conditions. The interactions are followed by circular dichrosim measurements, fluorescent intercalator displacement (FID) assays and molecular docking studies through MOE program. The free ligand and its Cu(II) and Zn(II) complexes stabilize predominantly antiparallel G-quadruplex form under antiparallel and no salt conditions. The binding constant (Kb) values calculated for the ligand and complexes are in the range of 1.9 x 105 M-1 to 4.2 x 107 M-1 under various conditions show the higher affinity of G-quadruplex conformations. The FID assay using thiazole orange with HTelo20 clearly depicts the G-quadruplex stabilization through strong binding mode. Also, all the three compounds dpyatriz, Cu-dpyatriz and Zn-dpyatriz show an intercalative mode of interaction with antiparallel G quadruplex DNA in molecular docking studies. The compounds show cytotoxicity with the IC50 values in the range of 50 – 90 nM, and antitelomerase activity in the range of 5 to 10 μM.

G4mismatch: Deep neural networks to predict G-quadruplex propensity based on G4-seq data

G-quadruplexes are non-B-DNA structures that form in the genome facilitated by Hoogsteen bonds between guanines in single or multiple strands of DNA. The functions of G-quadruplexes are linked to various molecular and disease phenotypes, and thus researchers are interested in measuring G-quadruplex formation genome-wide. Experimentally measuring G-quadruplexes is a long and laborious process. Computational prediction of G-quadruplex propensity from a given DNA sequence is thus a long-standing challenge. Unfortunately, despite the availability of high-throughput datasets measuring G-quadruplex propensity in the form of mismatch scores, extant methods to predict G-quadruplex formation either rely on small datasets or are based on domain-knowledge rules. We developed G4mismatch, a novel algorithm to accurately and efficiently predict G-quadruplex propensity for any genomic sequence. G4mismatch is based on a convolutional neural network trained on almost 400 millions human genomic loci measured in a single G4-seq experiment. When tested on sequences from a held-out chromosome, G4mismatch, the first method to predict mismatch scores genome-wide, achieved a Pearson correlation of over 0.8. When benchmarked on independent datasets derived from various animal species, G4mismatch trained on human data predicted G-quadruplex propensity genome-wide with high accuracy (Pearson correlations greater than 0.7). Moreover, when tested in detecting G-quadruplexes genome-wide using the predicted mismatch scores, G4mismatch achieved superior performance compared to extant methods. Last, we demonstrate the ability to deduce the mechanism behind G-quadruplex formation by unique visualization of the principles learned by the model.

A label-free ThT-assisted fluorescence detection strategy of alkaline phosphatase activity based on MnO2 nanosheets

Alkaline phosphatase (ALP) is a metalloenzyme, the level of which is clinically significant as an abnormality of ALP activity results in several diseases. In the present study, we introduced a MnO2 nanosheet-based assay for ALP detection employing the adsorption and reduction characteristics of G-rich DNA probes and ascorbic acid (AA), respectively. Ascorbic acid 2-phosphate (AAP) was utilized to act as a substrate for ALP which hydrolyzes AAP generating AA. In the absence of ALP, MnO2 nanosheets adsorb the DNA probe destructing the G-quadruplex formation and showing no fluorescence emission. On the contrary, being present in the reaction mixture ALP hydrolyzes AAP yielding AA, then the AA reduce the MnO2 nanosheets into Mn2+, hence, the probe is free to react with a dye, thioflavin T (ThT), and synthesizes ThT/G-quadruplex to spark high fluorescence intensity. Therefore, under optimized conditions (250 nM DNA probe, 8 μM ThT, 96 μg/mL MnO2 nanosheets, and 1 mM AAP) the sensitive and selective measurement of ALP activity can be achieved through the change of fluorescence intensity, with a linear range and a limit of detection of 0.1–5 U/L and 0.045 U/L. Our assay exhibited its potential to assess the ALP inhibitor when in an inhibition assay Na3VO4 inhibited ALP with an IC50 value of 0.137 mM and also was validated in clinical samples.

Label-free and highly sensitive fluorescent detection of bleomycin based on CRISPR-Cas12a and G-quadruplex-thioflavin T

CRISPR-Cas12a system opens up a new avenue for biosensing due to its flexible programmability and excellent target recognition accuracy. In this work, a novel label-free fluorescent biosensor for highly sensitive detection of bleomycin (BLM) was developed based on BLM-mediated inactivation of CRISPR-Cas12a and thioflavin T (ThT)-induced G-quadruplex formation. We designed a single-stranded DNA (ssDNA) probe containing the scission site of BLM as a DNA activator for Cas12a-crRNA. In the absence of BLM, the DNA activator binds to and activates the Cas12a, the activated CRISPR-Cas12a system trans-cleaves the ThT-induced G-quadruplex formation. Upon the addition of BLM, the DNA activator undergoes an irreversible cleavage event at the scission site via the oxidative effect of BLM with Fe(Ⅱ) as a cofactor. As a result, the trans-cleavage activity of Cas12a cannot be activated so the G-quadruplex-ThT complex remains intact, resulting in a significant increase of fluorescence signal. Attributing to the specific recognition of BLM and the excellent cleavage activity of Cas12a on DNA G-quadruplexes, the fluorescent biosensor exhibits high sensitivity and selectivity for detection of BLM in human serum samples, illustrating a promising tool for BLM assay in biomedical research.

Entropy-driven dynamic self-assembled DNA dendrimers for colorimetric detection of African swine fever virus.

Herein, we subtly engineered an amplified colorimetric biosensor for the cyclic detection of African swine fever virus DNA (ASFV-DNA), which associated the branched catalytic hairpin assembly (bCHA) amplification with G-quadruplex DNAzyme activity through triplex DNA formation. Firstly, a Y-shaped hairpin trimer was constructed for the dynamic self-assembly of DNA dendrimers. Then, in the presence of ASFV-DNA, the signal strand CP was opened, exposing the toehold regions, which would trigger the CHA cascade reaction between hairpin trimers. In the CHA cascade reaction, H1, H2, and H3 opened and bound in sequence, eventually forming the structure of DNA dendrimers. Subsequently, the obtained bCHA product was specifically recognized by the GGG repeat sequences of L1 and L2, then amplified by the synergistic effect of triplex DNA and the formation of asymmetric split G-quadruplex. Benefiting from the amplification properties of bCHA and the high peroxidase-like catalytic activity of asymmetrically split G-quadruplex DNAzymes, it could achieve effective colorimetric signal output in the presence of ASFV-DNA by means of triplex DNA formation. Under the optimal experimental conditions, this biosensor exhibited excellent sensitivity with a detection limit of 1.8pM. Further, it was applied to the content detection of simulated samples of African swine fever, and the recoveries were 98.9 ~ 103.2%. This method has the advantages of simple operation, good selectivity, and high sensitivity, which is expected to be used for highly sensitive detection of actual samples of African swine fever virus.

Inhibited complete folding of consecutive human telomeric G-quadruplexes.

Noncanonical DNA structures, termed G-quadruplexes, are present in human genomic DNA and are important elements in many DNA metabolic processes. Multiple sites in the human genome have G-rich DNA stretches able to support formation of several consecutive G-quadruplexes. One of those sites is the telomeric overhang region that has multiple repeats of TTAGGG and is tightly associated with both cancer and aging. We investigated the folding of consecutive G-quadruplexes in both potassium- and sodium-containing solutions using single-molecule FRET spectroscopy, circular dichroism, thermal melting and molecular dynamics simulations. Our observations show coexistence of partially and fully folded DNA, the latter consisting of consecutive G-quadruplexes. Following the folding process over hours in sodium-containing buffers revealed fast G-quadruplex folding but slow establishment of thermodynamic equilibrium. We find that full consecutive G-quadruplex formation is inhibited by the many DNA structures randomly nucleating on the DNA, some of which are off-path conformations that need to unfold to allow full folding. Our study allows describing consecutive G-quadruplex formation in both nonequilibrium and equilibrium conditions by a unified picture, where, due to the many possible DNA conformations, full folding with consecutive G-quadruplexes as beads on a string is not necessarily achieved.

G-quadruplex from precursor miR-1587 modulated its maturation and function

A certain proportion of pre-miRNAs, which contained potential G-quadruplex forming sequences, was found to act as a mediator to Dicer-mediated cleavage, and that the regulation of miRNA production and function may be achieved through the G-quadruplex structure. In this study, human precursor miR-1587 sequence was transfected after the incubation with different solution conditions (K+, TMPyP4, etc.). Firstly, the formation of G-quadruplex from precursor miR-1587 sequences was confirmed by CD and UV melting. The expression of miR-1587 level was then evaluated by Q-RT-PCR, and the results showed that the formation of G-quadruplex inhibited the miR-1587 maturation process, resulting in a reduced miR-1587 expression. Meanwhile the destabilization of G-quadruplex led to an increased miR-1587 expression by contrast. Then, the weakened inhibition of miR-1587 towards its target genes, such as TAGLN or NCOR1, was presented confirming by Q-RT-PCR and western blot. Molecular mechanism by dual-luciferase assays showed that the modulations of miR-1587 expression and function were due to the G-quadruplex structure transformation, but not the simple change of solution conditions. This study highlighted the importance of maintaining specific structures during miRNA biosynthesis and provided a way to alter the function of G-rich precursor miRNAs by modulating molecular conformation using ionic solutions or ligands.