Structure-switching aptasensors for sensitive detection of ochratoxin A.

Ochratoxin A (OTA) is a toxic metabolite commonly found in various foods and feedstuffs. Accurate and sensitive detection of OTA is needed for food safety and human health. Based on a common OTA-binding aptamer (OTABA), two structure-switching OTABAs, namely OTABA4 and OTABA3, were designed by configuring a split G-quadruplex and a split G-triplex, respectively, at the two ends of OTABA to construct aptasensors for the detection of OTA. The OTABA, G-quadruplex, and G-triplex all can capture the thioflavin T (ThT) probe, thereby enhancing the fluorescence intensity of ThT. Bonding with OTA could change the conformations of OTABA and G-quadruplex or G-triplex regions, resulting in the release of the captured ThT and diminution of its fluorescence intensity. Dual conformation changes in structure-switching OTABA synergistically amplified the fluorescence signal and improved the sensitivity of the aptasensor, especially for that with OTABA3. The detection limits of the OTABA4-ThT and OTABA3-ThT systems for OTA were 0.28 and 0.059 ng ml-1 , with a 1.4-fold and 6.7-fold higher sensitivity than that of the original OTABA-ThT system, respectively. They performed well in corn and peanut samples and met the requirements of the food safety inspections.

Telomere G-triplex lights up Thioflavin T for RNA detection: new wine in an old bottle.

Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT-based method. We believe this ht-G3/ThT-based label-free strategy could be widely applied for RNA detection.

Fluorescence method for kanamycin detection based on the conversion of G-triplex and G-quadruplex.

A versatile fluorescence scaffold was constructed by connecting a G-triplex sequence (G31) with G-rich DNA (aptamer of kanamycin) and using thioflavin T (ThT) as the fluorescent molecule. With the assistance of an aptamer, the G-quadruplex DNA structure was fabricated using G31 as three strands and the aptamer as the fourth strand. Due to the parallel planar morphology of the final products, which was favorable for ThT binding and which restricted the rotation of the aromatic rings of ThT, the fluorescence signal intensity of ThT was significantly enhanced. Because of the specific interaction of aptamer and kanamycin, in addition to the greater ability for kanamycin to bind with G-triplex than ThT, the conformation of G-quadruplex DNA was changed; in addition, ThT was dissociated from the aptamer-G31, and therefore a 'turn-on' to 'turn-off' detection principle was applied for kanamycin detection, which yielded reasonable sensitivity and selectivity. The detection range was 50-2000 nM, with a limit of detection of 1.05 nM. Our proposed method was thus successfully applied for kanamycin determination in pork, chicken, and beef samples, and satisfactory results were obtained.

An enzyme-free probe based on G-triplex assisted by silver nanocluster pairs for sensitive detection of microRNA-21.

A sensitive ratiometric fluorescence probe based on hybridization chain reaction (HCR) was constructed for sensitive detection of miRNA-21 by using G-triplex and silver nanocluster pairs (AgNC pairs) as an enzyme-free and label-free signal output group. miRNA-21 was used as the primer for the hybridization chain reaction of molecular beacon 1 (MB1) containing the locked G-triplex sequence and molecular beacon 2 (MB2) with intact AgNC pairs at the 5' and 3' end activation. The double-stranded product was obtained along with the opening of the G-triplex and the separation of the AgNC pairs. A detection limit of 67 pM and a linear detection range of 0.1-300 nM were obtained for miRNA-21 determination. The proposed strategy enabled the monitoring of miRNA-21 levels in at least three cell lines, indicating that it provided new ideas for detecting miRNA in real samples. MB1 and MB2 contained the locked G-triplex sequence and silver nanocluster pairs (AgNC pairs), respectively. In the presence of target, the hybridization chain reaction (HCR) between MB1 and MB2 was initiated. At the same time, the locked G-triplex was released and combined to the dye thioflavin T (THT) to increase fluorescence, while the separation of the AgNC pairs caused the fluorescence to decrease. The double-stranded (ds) DNA product was generated to form a ratiometric signal to be detected.

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection.

Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.

Sensitive colorimetric determination of microRNA let-7a through rolling circle amplification and a peroxidase-mimicking system composed of trimeric G-triplex and hemin DNAzyme.

The authors have incidentally found that the three tandem repeats of a 13-mer G-rich oligomer (with sequence 5'-TGG GAA GGG AGG G-3'; referred to as G3) can directly fold into a stable G3 trimer. The G3 trimer/hemin DNAzyme exhibits an about 3-fold higher peroxidase-mimicking activity compared to the conventional G3/hemin DNAzyme. Combining this finding with rolling circle amplification (RCA), a colorimetric assay was developed for sensitive and specific determination of microRNA. In this method, each cycle of RCA generates three catalytic units. This leads to a significant signal amplification of the RCA. Using let-7a as a model analyte, the colorimetric method (best performed at 420 nm) exhibits high sensitivity toward microRNA-let-7a with a 37 fM detection limit and an analytical range that covers 3 orders of magnitude. The method was applied to the determination of let-7a in some cell lysates. Graphical abstractThis G-triplex trimer-based rolling circle amplification (RCA) method can produce three catalytic units per RCA cycle, which can significantly improve the amplification efficiency of RCA.

A topological transition from bimolecular quadruplex to G-triplex/tri-G-quadruplex exhibited by truncated double repeats of human telomere.

Human telomeric G-rich sequences can fold back into various conformations depending upon the salt (Na+ or K+) at physiological pH. On the basis of results obtained by native PAGE electrophoresis, circular dichroism, and UV-melting experiments, we report here that truncated sequences of human telomere (d-GGGTTAGGG; GM9, d-AGGGTTAGGG; GM10, d-TAGGGTTAGGG; GM11) adopt a varied range of quadruplex conformations as a function of the cation present. By correlating CD and gel electrophoresis experiments; it was concluded that the GM9 oligonucleotide can self-associate to form a tetramer quadruplex (antiparallel; AP) in Na+ solution and a mixture of G-triplex (AP) or tri-G-quadruplex (parallel; P) along with a tetramer G-quadruplex structure (AP) in K+. The GM10 oligonucleotide formed a bimolecular G-quadruplex in both Na+ and K+ solutions, while GM11 associated to form a bimolecular G-quadruplex (AP) structure in Na+ solution and a mixture of bimolecular G-quadruplex (AP) and bimolecular G-quadruplex (P) along with parallel G-triplex or antiparallel tri-G-quadruplex in K+. All the UV-melting profiles, thermal difference spectra, and CD melting curves suggested the formation of a variety of G-quadruplex conformations by the DNA sequences studied in Na+ and K+ ions. Hypothetical models for different conformations adopted by these DNA molecules have also been proposed, which may further enhance our knowledge about the divergent topologies of guanine quadruplexes.

Discovery of the first dual G-triplex/G-quadruplex stabilizing compound: a new opportunity in the targeting of G-rich DNA structures?

BACKGROUND: Guanine-rich DNA motifs can form non-canonical structures known as G-quadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex. METHODS: Virtual screening was employed to identify a small set of putative G-triplex ligands. The G-triplex stabilizing properties of these compounds were analyzed by CD melting assay. DSC, non-denaturing gel electrophoresis, NMR and molecular modeling studies were performed to investigate the interaction between the selected compound 1 and G-rich DNA structures. Cytotoxic activity of 1 was evaluated by MTT cell proliferation assay. RESULTS: The experiments led to the identification of a promising hit that was shown to bind preferentially to G-triplex and parallel-stranded G-quadruplexes over duplex and antiparallel G-quadruplexes. Molecular modeling results suggested a partial end-stacking of 1 to the external G-triad/G-tetrads as a binding mode. Biological assays showed that 1 is endowed with cytotoxic effect on human osteosarcoma cells. CONCLUSIONS: A tandem application of virtual screening along with the experimental investigation was employed to discover a G-triplex-targeting ligand. Experiments revealed that the selected compound actually acts as a dual G-triplex/G-quadruplex stabilizer, thus stimulating further studies aimed at its optimization. GENERAL SIGNIFICANCE: The discovery of molecules able to bind and stabilize G-triplex structures is highly appealing, but their transient state makes challenging their recognition. These findings suggest that the identification of ligands with dual G-triplex/G-quadruplex stabilizing properties may represent a new route for the design of anticancer agents targeting the G-rich DNA structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Direct and single-molecule visualization of the solution-state structures of G-hairpin and G-triplex intermediates.

We present the direct and single-molecule visualization of the in-pathway intermediates of the G-quadruplex folding that have been inaccessible by any experimental method employed to date. Using DNA origami as a novel tool for the structural control and high-speed atomic force microscopy (HS-AFM) for direct visualization, we captured images of the unprecedented solution-state structures of a tetramolecular antiparallel and (3+1)-type G-quadruplex intermediates, such as G-hairpin and G-triplex, with nanometer precision. No such structural information was reported previously with any direct or indirect technique, solution or solid-state, single-molecule or bulk studies, and at any resolution. Based on our results, we proposed a folding mechanism of these G-quadruplexes.

Plug-and-play smart transistor bio-chips implementing point-of-care diagnosis of AMI with modified CRISPR/Cas12a system.

The point-of-care diagnosis of acute myocardial infarction (AMI), an extremely lethal disease with only a few hours of golden rescue time, is significant and urgently required. Here, we describe a plug-and-play carbon nanotube field effect transistor (CNT-FET) bio-chip supported with a smart portable readout for ultrasensitive and on-site testing of cardiac troponin I (cTnI), which is one of the most specific and valuable biomarkers of AMI. A modified clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system, featuring the G-triplex structured reporter, was first combined with the CNT-FET to realize non-nucleic acid detection. Such a unique CNT-FET biosensor achieved the high sensitivity (LOD: 0.33 fg/mL), which is expected to give timely warning in the early stage of myocardial injury. In addition, a bilayer gate dielectric consisting of Y2O3/HfO2, employed into the passivation process, enabled the high environmental stability and repeatability of CNT-FET. More importantly, the homemade compact chip readout forged a field-deployable cTnI analytical tool, realizing "plasma-to-answer" performance for AMI patients in point-of-care testing scenarios. The developed technology holds promise to help doctors make clinical decisions faster, especially in remote areas.

Label-free and ultrasensitive detection of environmental lead ions based on spatially localized DNA nanomachines driven by hyperbranched hybridization chain reaction.

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring.

Label-free and portable detection of HIV-DNA by a handheld luminometer.

BACKGROUND: The human immunodeficiency virus (HIV) remains a major worldwide health problem. Nowadays, many methods have been developed for quantitative detecting human immunodeficiency virus DNA (HIV-DNA), such as fluorescence and colorimetry. However, these methods still have the disadvantages of being expensive and requiring professional technicians. Early diagnosis of pathogens is increasingly dependent on portable instruments and simple point-of-care testing (POCT). Therefore, it is meaningful and necessary to develop portable and cheap methods for detecting disease markers. RESULTS: In this work, a label-free chemiluminescence (CL) method was developed for detecting HIV-DNA via a handheld luminometer. To achieve label-free target detection, the CL catalyst, G-triplex-hemin DNAzyme (G3-hemin DNAzyme), was in-situ assembled in the presence of HIV-DNA. For improving sensitivity, HIV-DNA induced the cyclic strand displacement reaction (SDR), which can form three G3-hemin DNAzymes in one cycle. So, the chemiluminescence reaction between luminol and H2O2 was highly effectively catalyzed, and the CL intensity was linearly related with the concentration of HIV-DNA in the range of 0.05-10 nM with a detection limit of 29.0 pM. Due to the high specificity of hairpin DNA, single-base mismatch can be discriminated, which ensured the specific detection of HIV-DNA. SIGNIFICANCE: In-situ formation of G3-hemin DNAzyme led to label-free and selective detection without complex synthesis and functionalization. Therefore, it offers a cheap, selective, sensitive and portable method for detecting disease-related genes, which is promising for POCT of clinical diagnosis in resource-limited settings.

G-triplex/hemin DNAzyme mediated colorimetric aptasensor for Escherichia coli O157:H7 detection based on exonuclease III-assisted amplification and aptamers-functionalized magnetic beads.

Escherichia coli O157: H7 (E. coli O157: H7) is one of the most common foodborne pathogens and is widespread in food and the environment. Thus, it is significant for rapidly detecting E. coli O157: H7. In this study, a colorimetric aptasensor based on aptamer-functionalized magnetic beads, exonuclease III (Exo III), and G-triplex/hemin was proposed for the detection of E. coli O157: H7. The functional hairpin HP was designed in the system, which includes two parts of a stem containing the G-triplex sequence and a tail complementary to cDNA. E. coli O157: H7 competed to bind the aptamer (Apt) in the Apt-cDNA complex to obtain cDNA. The cDNA then bound to the tail of HP to trigger Exo III digestion and release the single-stranded DNA containing the G-triplex sequence. G-triplex/hemin DNAzyme could catalyze TMB to produce visible color changes and detectable absorbance signals in the presence of H2O2. Based on the optimal conditions, E. coli O157: H7 could be detected down to 1.3 × 103 CFU/mL, with a wide linear range from 1.3 × 103 to 1.3 × 107 CFU/mL. This method had a distinguished ability to non-target bacteria, which showed good specificity. In addition, the system was successfully applied to detect E. coli O157: H7 in milk samples.

Construction of a novel "self-regenerative" electrochemical biosensor based on metal-organic frameworks and its application to the detection of Mycoplasma ovine pneumonia.

This study aimed to prepare a novel "self-regenerative" electrochemical biosensor by successively modifying gold nanoparticles, four-arm polyethylene glycol-NH2, and NH2-MIL-53 (Al) (MOF) on the glassy carbon electrode interface. A hairpin G-triplex-mediated DNA (G3 probe) as a part of the mycoplasma ovine pneumonia (MO) gene was loosely adsorbed to MOF. Based on the mechanism of hybridization induction, the G3 probe could effectively detach from the MOF only after introducing the target DNA. Subsequently, its guanine-rich nucleic acid sequences were exposed to solution of methylene blue. As a result, the diffusion current of the sensor system showed a sharp decline. The developed biosensor showed excellent selectivity, and the concentration of target DNA exhibited a good correlation in the range 10-10 to 10-6 M with a detection limit of 1.00 pM (S/N = 3), even in 10% goat serum. Most interestingly, this biosensor interface automatically started the regeneration program. Moreover, regeneration could be effectively achieved at least seven times, and the recovery rate of the electrode interface and sensing efficiency was up to 90%. Additionally, this platform could be used for other clinical assays in various systems by simply changing the DNA sequence of the probe.

Label-free fluorescence detection of alkaline phosphatase activity using a G-triplex based dumbbell-shaped probe.

Based on the fluorescence enhancement property of the G-triplex (G3)-Thioflavin T (ThT) complex, a fluorescent biosensor was successfully constructed for detection of ALP using a G3-based dumbbell-shaped probe (DP). In this work, calf intestinal ALP (CIP) can act on the 5'-terminal phosphate of DP, thereby regulating the subsequent DNA ligation reaction and enzyme cleavage of the DP nick. When the DP is digested by exonuclease, the released G3 can bind to ThT, resulting in enhanced fluorescence signal. The linear range of the sensor for CIP detection is 0.00002-0.002 U/µL, and the detection limit is 1.8 × 10-5 U/µL. The proposed method has the advantages of simplicity, no fluorophore labeling, and low cost, which was successfully applied to the screening of enzyme inhibitors and ALP determination in human serum samples. To the best of our knowledge, this is the first report of a biosensor using G3-ThT as the signal indicator for ALP detection, which should promote the further exploitation of applying G3-ThT complex in the field of various targets recognition and analysis.

The effect of adjacent double-strand DNA on the G-triplex-ThT complex fluorescence intensity enhancement and its application in TNOS and Hg2+ detection.

In this paper, we have found that adjacent double-strand DNA (dsDNA) can enhance the fluorescence intensity of the G-triplex (G31)-thioflavin T (ThT) complex. By connecting the newly formed dsDNA with the G31 sequence, terminator nopaline synthase (TNOS) gene and Hg2+ were detected. The intermolecular duplex hybridization (e.g., TNOS gene and its complementary DNA), or the intramolecular mismatched thymine (T)-Hg2+-T pairs induced the formation of dsDNA with planar morphology, which resulted in the increased binding capacity of adjacent G31 to ThT, as well as the enhanced fluorescence intensity of G31-ThT complex. A versatile 'turn-on' fluorescence scaffold was developed for discriminating transgenic and non-transgenic soybeans and detecting Hg2+ in lake water.

A G-triplex and G-quadruplex concatemer-enhanced fluorescence probe coupled with hybridization chain reaction for ultrasensitive aptasensing of ochratoxin A.

Ochratoxin A (OTA), a typical mycotoxin contaminant found in various agricultural products and foods, poses a serious threat to human health. In this study, an aptasensor based on a novel fluorescence probe comprising a G-rich DNA sequence (G43) and thioflavin T (ThT) was designed via hybridization chain reaction (HCR) for the ultrasensitive detection of OTA. G43 is a concatemer of G-quadruplex and G-triplex (a G-quadruplex-like structure with one G-quartet removed), which can drastically enhance the fluorescence intensity of ThT. For this strategy to work, the OTA aptamer is pro-locked in a hairpin structure, denoted "hairpin-locked aptamer" (HL-Apt). OTA binds to HL-Apt, opens the hairpin structure, releases the trigger sequence, and initiates the HCR reaction to form a long DNA duplex and numerous side chains. The side chains combine entirely with the complementary DNA and liberate the pro-locked G43 DNA, dramatically enhancing the intensity of the ThT fluorescence signal. The fluorescence intensity correlates linearly with the OTA concentration between 0.02 and 2.00 ng mL-1, and the method has a detection limit of 0.008 ng mL-1. The developed aptasensor was used to detect OTA in foodstuffs with satisfactory results.

Improving efficiency of entropy-driven DNA amplification biosensing through producing two label-free signal strands in one cycle.

Entropy-driven circuits (EDC) provides one isothermal and non-enzymatic signal amplification strategy. But the efficiency of EDC signal amplification is not enough high because only one single strand can be produced in each cycle of the typical EDC system. In this work, we proposed one strategy to improve the amplification efficiency of EDC-based biosensing. In this strategy, two signal strands were produced in one cycle. The G-triplex (G3)-forming sequence was used as signal strand, and the G3/thioflavin T (G3/ThT) was used as label-free fluorescence reporter in this EDC-based biosensing. The detection limit of this method was estimated to be 3.4 pM for target DNA, which was about 10 times lower than that of the conventional EDC method. Furthermore, the response time was shortened from more than 1 h-0.5 h. In a word, one enzyme-free and label-free EDC strategy was proposed to construct an efficient nucleic acid biosensing platform.

A generic and non-enzymatic electrochemical biosensor integrated molecular beacon-like catalyzed hairpin assembly circuit with MOF@Au@G-triplex/hemin nanozyme for ultrasensitive detection of miR-721.

MicroRNAs (miRNAs) have been extensively studied as circulating biomarkers for early diagnosis and prognosis of many human diseases. However, it has been found challenging to accurately detect and quantify the trace amounts of miRNAs in biological samples. Herein, we propose a generic and non-enzymatic electrochemical strategy integrated molecular beacon-like catalyzed hairpin assembly (mCHA) circuit with MOF@Au@G-triplex/hemin nanozyme for ultrasensitive detection of miR-721 (a novel diagnostic biomarker of acute myocarditis). Nitro-functionalized MIL-101 functions as an ideal nanocatalyst and nanocarrier to facilitate efficient immobilization of G-triplex/hemin DNAzyme, to form signal probes. Tetrahedral nanostructured DNA probes self-assemble onto the Au nanoparticles/proton-functionalized graphitic carbon nitride nanosheets films, to fabricate a coordinated sensing interface. A mCHA circuit is designed to convert and amplify each target to DNA duplexes, which cause signal probes anchored on the sensing interface and produce an enhanced electrochemical signal. With the assistance of the mCHA circuit, double-amplified nanozymes and sensing interface for signal amplification, this biosensor had a low detection limit of 0.25 fM and high specificity. The proposed biosensor has been successfully used in miR-721 detection in real biological samples, which provided a promising potential method for acute myocarditis early diagnosis.

A sensitive aptasensor based on rolling circle amplification and G-rich ssDNA/terbium (III) luminescence enhancement for ofloxacin detection in food.

As the light-harvesting "antenna", G-rich oligonucleotides (such as the G-quadruplex) can interact with lanthanide (III) to bring a luminescent enhancement response. In this study, phenomenon of luminescent enhancement of G-triplex/terbium (III) (G3/Tb3+) and interaction between G3 and Tb3+ were first reported and characterized. Based on G3/Tb3+ luminescence, a label-free aptasensor for the detection of ofloxacin (OFL) residues in the food was developed. The OFL triggered the action of rolling circle amplification (RCA) allowed for the amplification product of G3-forming sequences in the single-stranded DNA, which promoted the conformational transition of the G3/Tb3+ complexes once the addition of Tb3+. Under the optimal conditions, the logarithmic correlation between the G3/Tb3+ luminescence intensity and the concentration of OFL was found to be linear in the range of 5-1000 pmol L-1 (R2 = 0.9949). The limit of detection was 0.18 pmol L-1 (3σ/slope). Additionally, the good recoveries of 90.19-108.89 % and the relative standard deviations values of 0.59-5.87 % were obtained in the application of the aptasensor detecting OFL in the practical samples. These results confirmed that the present aptasensor has a good analytical performance and bright prospect for detecting ofloxacin residues in food.