G-quadruplex DNAzyme Research Articles

Target-triggered assembly of functional G-quadruplex DNAzyme nanowires for sensitive detection of miRNA in lung tissues

MicroRNAs (miRNAs) are important biomarkers for multiple diseases including cancers, neurodegenerative diseases, and cardiovascular diseases. Accurate detection of miRNAs is imperative for early diagnosis and biomedical research. Herein, we demonstrate target-triggered assembly of G-quadruplex DNAzyme nanowires for sensitive detection of miRNAs in lung tissues by integrating primer generation-mediated rolling circle amplification (PG-RCA) with primer exchange reaction (PER). Target miRNA hybridizes with the circular template to initiate PG-RCA, producing numerous primers. The obtained primers then react with the catalytic hairpin DNA template to trigger PER, producing the polymer G-quadruplex DNAzyme nanowires. Eventually, thioflavin T (ThT) as a fluorescence indicator selectively interacts with the G-quadruplex DNAzyme to generate an amplified signal. This assay is capable of detecting miRNA over a large dynamic range (up to 6 orders of magnitude) and it exhibits high sensitivity with a limit of detection (LOD) of 61.34 aM. Moreover, this assay displays excellent specificity and it can distinguish even a single-base mutation in miR-486–5p. Furthermore, this method can quantify the miR-486–5p concentration in different types of cells, and it enables the identification of differentially expressed miR-486–5p in lung tissues of normal persons and nonsmall-cell lung carcinoma (NSCLC) patients, holding promising applications in biomedical analysis and cancer prognosis.

Sensitive colorimetric detection of miRNA-155 via G-quadruplex DNAzyme decorated spherical nucleic acid.

Rapid and sensitive detection of biomarkers enables monitoring patients' health status and can enhance the early diagnosis of deadly diseases. In this work, we have developed a new colorimetric platform based on spherical nucleic acid (SNA) and G-quadruplex DNAzymes for the identification of specific miRNAs. The simple hybridization between the target miRNA and two capture probes (capture probe 1 located at AuNP surface and free capture probe 2) is the working principle of this biosensor. The hybridization and duplex formation among probes and miRNAs led to a significant decrease in the intensity of color change. A linear relationship between the decrease of colorimetric signal and the amount of target molecules was witnessed from 1 to 100nM for miRNA-155. Using this method, we were able to detect concentrations of miRNA-155 as low as 0.7nM. Furthermore, the proposed sensing platform can be utilized profitably to detect miRNA-155 in real human serum samples. We further investigated the applicability of the proposed method in a microfluidic system which displayed promising results. In this project, A G-quadruplex based SNAzyme was constructed to provide a fast and simple colorimetric method for miRNA detection. The SNAzyme actually employed as both target recognition element and catalytic nano labels for colorimetric detection.

Target-induced tripedal G-quadruplex DNAzyme for multicolor visual point-of-care testing of biomarkers using Au nanorods-decorated electrospun nanofibrous films

A target-induced tripedal G-quadruplex DNAzyme (TGD) for visual point-of-care testing (POCT) of PSA proteins by using solid Au nanorods (Au NRs)-decorated electrospun nanofibrous films was constructed. The TGD was formed by the recycling amplification cascades of target-triggered catalytic hairpin assembly (CHA), which exhibited superior horseradish peroxidase (HRP) mimicking activity to facilitate the degradation of H2O2 into hydroxyl radicals (·OH). The ·OH oxidized the 3,3′,5,5′-tetramethylbenzidine (TMB) into TMB+. Then, the TMB+ was converted into TMB2+ in the acidic environment, which can etch the Au NRs for further promoting the blue shift of their longitudinal surface plasmon resonance (LSPR) absorption peak. Thereby, with the increasing PSA concentrations, the proposed strategy presented palpable color variations from green, cyan, violet, and red by naked eyes. Significantly, the cutoff values near 4.0 and 10.0 ng/mL of PSA in human blood serum could be discriminated with naked eyes, which could provide a semi-quantitative process for the early diagnosis and prognosis evaluation. In addition, this portable, and label-free TDG biosensor exhibited remarkable improvements in sensitivity with a low detection limit of 61.1 fg/mL for PSA protein. This strategy showed the great versatility and potential for the visual biomedical detection with portable type and early diagnosis of diseases.

Self-Assembly of DNA Tiles with G-Quadruplex DNAzyme Catalytic Activity for Sensing Applications.

DNA tiles form through self-assembly of a small number of DNA strands that interact through basic repeated interactions, allowing the growth of nanoscale structures seeded by molecular inputs. If an approach for catalytic signal amplification can be integrated into the resultant nanostructure, then one can anticipate biosensing or diagnostic applications mediated by DNA tile self-assembly. Here, two-dimensional DNA tiles with split quadruplexes were designed as diagnostic tools for nucleic acid sensing without the use of protein enzymes. The presence of a target sequence leads to formation of extended microscale structures with arrayed multiple G-quadruplexes across the tile plane, with catalytic activity coupled to a colorimetric reporter. Such a mechanism has potential for low-cost signal amplification using unmodified DNA without the use of protein enzymes for biosensing.

Responsive Cysteine-Lighted Silver Nanoclusters Regulated by Highly Catalytic G-Quadruplex DNAzyme for Ultrasensitive Detection of Salmonella Typhimurium

Responsive Cysteine-Lighted Silver Nanoclusters Regulated by Highly Catalytic G-Quadruplex DNAzyme for Ultrasensitive Detection of Salmonella Typhimurium

A novel and label-free electrochemical aptasensor based on exonuclease III and G-quadruplex DNAzyme for sensitive and selective detection of metronidazole

A novel and label-free electrochemical aptasensor based on exonuclease III (Exo III) and G-quadruplex DNAzyme has been first designed to detect metronidazole (MNZ) sensitively and selectively in this paper. Capture probe (CP), helper probe (HP) and aptamer (Apt) hybridize to form double-stranded DNA (dsDNA) structure with a blunt 3′-terminus, resulting in the guanine (G)-rich CP being enclosed in the dsDNA, followed by specific digestion of the 3′-terminus by Exo III to expose the G-rich sequence. At this time, CP can bind with the added hemin to form G-quadruplex DNAzyme which catalyzes the redox reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2, generating a strong current signal. However, in the presence of MNZ, the binding of MNZ and aptamer breaks the original blunt 3′-terminus of the dsDNA structure, blocking the subsequent series of reactions, and only a weak current signal can be observed. Under the optimal conditions, the aptasensor showed excellent sensitivity and specificity for the detection of MNZ with a wide linear range from 10 pM to 500 nM and a detection limit as low as 3.0 pM (S/N = 3). The aptasensor constructed based on MNZ aptamer can effectively avoid the interference of other nitro compounds and antibiotics to a certain extent. Compared with other specific recognition elements, the aptasensor possesses the advantages of strong specificity, simple preparation and low cost. Moreover, the proposed aptasensor has been successfully applied to the detection of MNZ in tap water and honey samples.

Ferrocene-Doped Polystyrene Nanoenzyme and DNAzyme Cocatalytic SERS Quantitative Assay of Ultratrace Pb2.

A new, stable and high-catalytic activity ferrocene-doped polystyrene nanosphere (PNFer) sol was prepared by the hydrogel procedure and characterized by electron microscopy and molecular spectroscopy. Results show that the nanosol exhibits excellent catalysis of the new indicator nanoreaction between AgNO3 and sodium formate to generate nanosilver with strong surface-enhanced Raman scattering (SERS), resonance Rayleigh scattering (RRS) and surface plasmon resonance absorption (Abs) trimode molecular spectral signals. This new nanocatalytic amplification trimode indicator reaction was coupled with the G-quadruplex DNAzyme catalytic amplification of Pb2+ aptamer to fabricate a new SERS quantitative/RRS/Abs assay platform for the determination of ultratrace amounts of Pb2+. The Pb2+ content in water samples was analyzed with satisfactory results.

Electrochemical microRNA detection based on catalytic deposition of G-quadruplex DNAzyme in nanochannels

Electrochemical microRNA detection based on catalytic deposition of G-quadruplex DNAzyme in nanochannels

Visible light-driven i-motif-based DNAzymes

DNA foldings provide variant possibilities to develop DNAzymes with remarkable catalytic performance. In spite of fruitful reports on G-quadruplex DNAzymes, four-stranded cytosine-rich i-motifs have not been explored as the potential skeletons of DNAzymes. In this work, we developed a visible light-driven DNAzyme based on human telomeric i-motifs using a natural photosensitizer of hypericin (Hyp) as the cofactor and dissolved oxygen as the oxidant source. The i-motif folding in acidic solution caused the distal thymine overhangs at the 3′ and 5′ ends to approach each other to provide a favorable binding site for Hyp via an interaction of fully complementary hydrogen bonding. However, the i-motifs without the distal overhangs or with the inappropriate overhang length and the base identity exhibited no binding with Hyp. The binding event converted Hyp from the fully dark state to the emissive state under visible light illumination. Subsequently, the excited Hyp had an opportunity to transfer energy to dissolved oxygen. Resultantly, singlet oxygen (1O2) was generated to initiate the substrate oxidation. The catalytic performance of the DNAzyme can be improved using a long-lived mediator. Our developed i-motif-based DNAzyme can be driven by almost the whole range of visible lights, suggesting broad applications in the photocatalytic fields, for example, as an alternative strategy in developing biodevices.

Visual detection of aflatoxin B1 and zearalenone via activating a new catalytic reaction of "naked" DNAzyme.

Here, we have found for the first time that the catalytic activity of "naked" DNAzyme, a single-stranded G-quadruplex DNAzyme (S.DNAzyme), can be modulated by aflatoxin B1 (AFB1) and zearalenone (ZEN). In fact, S.DNAzyme can mimic the activity of horseradish peroxidase to perform oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to colored ABTS˙+ (dark green). This catalytic activity can be inhibited upon addition of AFB1, leading to color fade of the solution. But with ZEN, the solution color will change to a yellowish-brown or yellow color. Thus, we have exploited this finding to achieve label-free, naked-eye detection of AFB1 or ZEN, and have explored the possible detection mechanisms. This approach is able to detect AFB1 and ZEN concentrations as low as 0.18 μM and 0.29 μM, respectively. Even in real samples, the limit of detection values of these two analytes are lower than 3 μM. Notably, S.DNAzyme cross-reacted with three other tested aflatoxins, including aflatoxin B2, aflatoxin G1 and aflatoxin M1, revealing the potential for a broadly applicable method to identify the family of aflatoxins. The AFB1- or ZEN-triggered new catalytic reaction between "naked" DNAzyme and ABTS may offer new opportunities for on-site visual detection of mycotoxins.

An entropy-driven signal-off DNA circuit for label-free, visual detection of small molecules with enhanced accuracy.

An entropy-driven DNA circuit offers an efficient means of sensitive analyte detection with signal amplification. In this article, we rationally engineered an aptamer-based entropy-driven signal-off DNA circuit for colorimetric detection of small molecules. The proposed signal-off DNA circuit is activated by target small molecule binding to drive the collapse of G-quadruplex DNAzyme, accompanied by the colour change of the detection solution from dark blue to light blue. Entropy-driven recycling hybridization significantly magnified the input signal of the target small molecule. Such an assay enables naked-eye detection of adenosine triphosphate and oxytetracycline at concentrations as low as 0.5 μM and 1 μM respectively. Moreover, when compared with the signal-on DNA circuit, the entropy-driven signal-off DNA circuit for colorimetric detection has two advantages. Firstly, unlike in the signal-on DNA circuit, the unavoidable formation of waste complexes in the absence of a target in the signal-off DNA circuit has no influence on target detection performance as its background signal is only determined by the substrate complex. Secondly, the signal-on DNA circuit cannot distinguish false-positive signals generated by invasive catalysts (e.g., HRP, serum, Fe3O4), while the signal-off DNA circuit can distinguish those signals as undesired signals. Overall, the signal-off DNA circuit affords a novel strategy for sensitive and accurate detection of small molecules.

Target biorecognition-triggered assembly of a G-quadruplex DNAzyme-decorated nanotree for the convenient and ultrasensitive detection of antibiotic residues

The abuse of kanamycin (Kana) in many fields has led to increasing antibiotic pollution problems and serious threats to public health. Therefore, determining how to develop methods to realize the convenient detection of antibiotics in complicated environmental matrices is highly desirable. In this study, we utilized a target biorecognition-triggered hybridization chain reaction (HCR) assembly of a G-quadruplex DNAzyme (G-DNAzyme)-decorated nanotree to develop a novel homogeneous colorimetric biosensing method for the convenient and ultrasensitive detection of Kana antibiotic residues in real samples. Through the designed aptamer-recognition reaction, an Mg2+-dependent DNAzyme (MNAzyme) strand can be liberated. Thus, its catalyzed cleavage of the hairpin substrates anchored at a DNA nanowire will cause the assembled formation of an HCR-initiator; this process can be greatly amplified by the exonuclease III-assisted target recycling and the MNAzyme-catalyzed release of another MNAzyme strand. Based on the DNA-nanowire-accelerated HCR assembly of many G-DNAzyme-decorated DNA duplexes on the two sides of the nanowire, a DNA nanotree decorated by numerous G-DNAzymes will form to realize the ultrasensitive colorimetric signal output. Under the optimal conditions, this method exhibited a wide five-order-of-magnitude linear range and a very low detection limit of 28 fg mL−1. In addition, excellent selectivity, repeatability, and reliability were also demonstrated for this homogeneous bioassay method. These unique features along with its automatic manipulation and low assay cost show promise for practical applications.

Cooperative In Situ Assembly of G-Quadruplex DNAzyme Nanowires for One-Step Sensing of CpG Methylation in Human Genomes.

CpG methylation is one the most predominant epigenetic modification that has been recognized as a molecular-level biomarker for various human diseases. Taking advantage of methylation-dependent cleavage and encoding flexibility in nucleic acid functions and structures, we demonstrate the cooperative in situ assembly of G-quadruplex DNAzyme nanowires for one-step sensing of CpG methylation in human genomes. This nanodevice displays good specificity and high sensitivity with a limit of detection (LOD) of 0.565 aM in vitro and 1 cell in vivo. It can distinguish 0.001% CpG methylation level from excess unmethylated DNA, quantify different CpG methylation targets from diverse human cancer cells, and even discriminate CpG methylation expressions between lung tumor and precancerous tissues. Importantly, this nanodevice can be performed isothermally in one step within 2 h in a label-free manner without any bisulfite conversion, fluorescence tagging, and PCR amplification process, providing a new platform for genomic methylation-related clinical diagnosis and biomedical research.

Padlock Probe-Based Generation of DNAzymes for the Colorimetric Detection of Antibiotic Resistance Genes.

The increasing emergence of multidrug- and pan-resistant pathogens requires rapid and cost-efficient diagnostic tools to contain their further spread in healthcare facilities and the environment. The currently established diagnostic technologies are of limited utility for efficient infection control measures because they are either cultivation-based and time-consuming or require sophisticated assays that are expensive. Furthermore, infectious diseases are unfortunately most problematic in countries with low-resource settings in their healthcare systems. In this study, we developed a cost-efficient detection technology that uses G-quadruplex DNAzymes to convert a chromogenic substrate resulting in a color change in the presence of antibiotic resistance genes. The assay is based on padlock probes capable of high-multiplex reactions and targets 27 clinically relevant antibiotic resistance genes associated with sepsis. In addition to an experimental proof-of-principle using synthetic target DNA, the assay was evaluated with multidrug-resistant clinical isolates.

Smartphone-assisted colorimetric detection of BRCA-1 gene based on catalytic hairpin assembly amplification and G-quadruplex DNAzyme

• An enzyme-free colorimetric biosensor for sensitive detection of BRCA-1 gene is developed. • The target-triggered CHA and the peroxidase-like activity of G-quadruplex DNAzyme are used for signal amplification. • The colorimetric biosensor exhibits a high sensitivity toward target DNA with a detection limit of 10 pM. • The proposed strategy has a great potential application in genetic analysis and clinical diagnosis. In this study, we develop an enzyme-free colorimetric biosensor for the determination of breast cancer susceptibility gene (BRCA-1), which integrates catalytic hairpin assembly (CHA) signal amplification with the peroxidase-like activity of G-quadruplex DNAzyme. With the addition of BRCA-1 gene, the hairpin probes (H1 and H2) are able to generate numerous duplex DNA assemblies with liberated G-quadruplex forming sequences. After binding with cofactor hemin, the G-quadruplex forming sequences fold into G-quadruplex DNAzymes, which effectively catalyze the H 2 O 2 -mediated oxidation of 2,2’-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) to free-radical cation (ABTS •+ ), accompanied by a color transition from colorless to green. The colorimetric readout can be recorded using a smartphone camera or measured by UV-vis spectrophotometer. The developed colorimetric biosensor can detect target DNA as low as 10 pM with a linear range from 50 pM to 200 nM. Meanwhile, the colorimetric biosensing platform allows the determination of target DNA at a concentration as low as 0.2 nM through smartphone applications. In addition, the colorimetric biosensor shows excellent selectivity and acceptable applicability for the detection of BRCA-1 gene in human serum samples with satisfactory recoveries of 93.47–104.14% and RSDs of 6.89–8.24%, which might hold great application potential in genetic analysis and clinical diagnosis.

Label-Free Colorimetric Method for Detection of Vibrio parahaemolyticus by Trimming the G-Quadruplex DNAzyme with CRISPR/Cas12a.

Vibrio parahaemolyticus (V. parahaemolyticus), which may cause gastrointestinal disorders in humans, is a pathogen commonly found in seafood. There are many methods for detecting V. parahaemolyticus, yet they have some shortcomings, such as high cost, labor-intensiveness, and complicated operation, which are impractical for resource-limited settings. Herein, we present a sequence-specific, label-free, and colorimetric method for visual detection of V. parahaemolyticus. This method utilizes CRISPR/Cas12a to specifically recognize the loop-mediated isothermal amplification (LAMP) products for further trans-cleaving the G-quadruplex DNAzyme and depriving its peroxidase-mimicking activity. In this way, the results can be directly observed with the naked eyes via the color development of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS2-), which displays colorless for positive samples while green for target-free samples. We term such Cas12a-crRNA preventing ABTS2- from developing color by trimming the G-quadruplex DNAzyme as Cascade. The proposed method can detect 9.8 CFU (per reaction) of pure cultured V. parahaemolyticus, and the sensitivity is comparable to real-time LAMP. It has been applied for practical use and showed the capability to detect 6.1 × 102 CFU/mL V. parahaemolyticus in shrimp samples. Based on this, the newly established Cascade method can be employed as a universal biosensing strategy for pathogenic bacterial testing in the field.

Dual CHA-mediated high-efficient formation of a tripedal DNA walker for constructing a novel proteinase-free dual-mode biosensing strategy

DNA walkers have been recognized as a type of powerful signal amplification tool for biosensors, but how to adopt a proper strategy to increase their amplification efficiency is still highly desirable. Herein we design a dual-catalytic hairpin assembly (CHA)-mediated strategy for the high-efficient formation of a tripedal Mg2+-dependent DNAzyme (MNAzyme)-DNA walker, and thus develop a novel proteinase-free dual-mode biosensing method for the kanamycin (Kana) antibiotic assay. The first CHA is initiated by a target-biorecognition reaction, which can produce the DNA walker and also induce the target recycling. The second CHA is initiated by a special base sequence designed as a one-half substrate of the MNAzyme. Upon the first CHA-triggered DNA walking at a magnetic bead (MB) track, this “pseudo-target” sequence can be released to induce another CHA-cycle for the formation of the same DNA walker. Meanwhile, the other one-half substrate strand exposed on the MB surface will trigger the quantitative hybridization chain reaction (HCR)-assembly of a G-quadruplex DNAzyme (G-DNAzyme)-enriched double-stranded DNA polymer. So the enzymatic reaction of G-DNAzymes enabled the convenient colorimetric and photoelectrochemical dual-mode signal transduction of the method. Due to the dual-CHA facilitation to the tripedal and three-dimensional DNA walking and synergetic signal amplification of HCR, this method exhibits very low detection limits of 9.4 and 0.55 fg mL−1, respectively. In combination with its wide linear range, automated manipulation, and excellent selectivity, repeatability and reliability, the proposed method is expected to be used for the convenient semiquantitative screening and accurate determination of possible antibiotic residues in complicated matrices.

An origami paper-based peptide nucleic acid device coupled with label-free DNAzyme probe hybridization chain reaction for prostate cancer molecular screening test

Prostate cancer associated 3 (PCA3) assay has been used to improve prostate cancer diagnosis and reduce unnecessary biopsies. In this work, we successfully developed a new PCA3 assay on an origami paper-based peptide nucleic acid device (oPAD). The PCA3 oPAD comprises an acrylic cassette and shutter slides to facilitate the molecular reaction and liquid control occurring on the paper surface. To quantify PCA3, a pyrrolidinyl peptide nucleic acid (acpcPNA) was immobilized onto the aldehyde-modified oPAD surface as a selective capture probe. A G-quadruplex (GQD) DNAzyme reporter probe was designed so that the PCA3 gene target binding triggered the hybridization chain reaction of the reporter probe, resulting in the accumulation of the GQD on the oPAD. The peroxidase activity of the GQD-hemin generated a deep green color of the oxidized ABTS substrate. Image analyses were performed in Adobe Photoshop CS6. The proposed oPAD was successfully applied in PCA3 detection ranges of 1–5 μM (r2 = 0.982) with a limit of detection of 0.5 μM. Our proposed oPAD was demonstrated to measure PCA3 samples in both urine matrix and human cancer cell lines. The results reveal the great potential of our origami paper-based platform to be an alternative approach for facile, rapid, and low-cost detection of PCA3 in real samples.

A DNAzyme-catalyzed label-free aptasensor based on multifunctional dendrimer-like DNA assembly for sensitive detection of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is an important malign tumor marker. In this study, a simple, label-free and antibody-free aptasensor was fabricated based on a multifunctional dendrimer-like DNA nanoassembly. The DNA nanoassembly was embedded with multiple G-quadruplex DNAzyme motifs and a hanging CEA aptamer motif. It was prepared from short DNA sequences by autonomous-assembly. The aptasensor was prepared simply by self-assembly of a capture DNA (cpDNA) on a gold electrode, followed by hybridization with a CEA aptamer (AptGAC-P). CEA as a model target was detected through competitive binding of CEA with AptGAC-P, exposing cpDNA to bind with the DNA nanoassembly. The detection process only contains 2 incubation steps. The high load of G-quadruplex DNAzyme motifs and their catalytic activity resulted in an amplified and label-free differential pulse voltammetry (DPV) electrochemical signal. The peak current correlated linearly with the CEA concentration, with a linear range of 2–45 ng mL−1, and an LOD value of 0.24 ng mL−1. The aptasensor showed high specificity and reproducibility, and retained 96.5% of detection signal intensities after 31 days of storage. The recovery rates for spiked CEA in human serum were within 100 ± 5%, and the coincidence rates for clinical human serum samples with ELISA kits were 80.7–111%. Conceivably, possessing simplicity, sensitivity, reproducibility, storage stability, and accuracy, the aptasensor should be a very prominent and applicable tool for clinical CEA detection and cancer diagnosis, and is promisingly applicable as a platform for detecting other targets of interests.

A visual cardiovascular biomarker detection strategy based on distance as readout by the coffee-ring effect on microfluidic paper

We first reported a novel visualized instrument-free signal conversion method on paper for cardiovascular biomarker detection based on the coffee-ring effect (CRE). Our strategy was based on the principle that cTnI was captured by DNA probe decorated detection antibody to form sandwich complex, which could hybridize with H1 probes to form G-quadruplex DNAzyme. As a peroxidase mimic, G-quadruplex DNAzyme could catalyze colorimetric reaction and generate colored products, which were converted to the end edge of the paper by evaporation-driven flow, forming a visible colored band. Hence, the resulted colored signals were converted to distance signal by CRE, so that the quantification of cTnI could be measured easily only by a ruler. Our results demonstrated that the length of the colored bands was proportional to the concentration of cTnI, with a limit of detection of 1.0 pg/mL. Furthermore, this strategy not only showed advanced performance to discriminate cTnI from complex samples but also exhibited high accuracy with standard method in real sample. Hence, these results demonstrated that this method established a promising equipment-free and visualized platform for detecting various kinds of genetic diseases and biomarkers, further promoting the development of point-of-care testing (POCT) in biomedicine.