Assistant deoxyribonucleic acid recycling with Zn and molecular beacon for electrochemical detection of deoxyribonucleic acid via target-triggered assembly of mutated DNAzyme

A novel enzyme-free amplification strategy was designed for sensitive electrochemical detection of deoxyribonucleic acid (DNA) based on Zn²⁺ assistant DNA recycling via target-triggered assembly of mutated DNAzyme. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme first hybridized and then cleaved the MB in the presence of cofactor Zn²⁺. After cleavage, the MB was cleaved into two pieces and the ferrocene (Fc) labeled piece dissociated from the gold electrode, thus obviously decreasing the Fc signal and forming a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles to trigger the cleavage of many MB substrates. Therefore, the peak current of Fc dramatically decreased to approximately zero. The strategy showed a detection limit at 35 fM levels, which was about 2 orders of magnitude lower than that of the conventional hybridization without Zn²⁺-based amplification. The Zn²⁺ assistant DNA recycling offers a versatile platform for DNA detection in a cost-effective manner, and has a promising application in clinical diagnosis.     

DNAzyme‐Based Probes for Telomerase Detection in Early‐Stage Cancer Diagnosis

Human telomerase is a polymerase enzyme that adds tandem repeats of DNA (TTAGGG) in the telomeric region to the ends of chromosomes. Since telomerase can be detected in immortalized, but not normal, somatic cells, it has been considered a selective target for cancer chemotherapy. Here, we describe a DNAzyme‐based probe to detect the presence of telomerase in cell lysates. Telomerase elongates the primer site on the probe. Subsequent addition of the Pb^II cofactor activates the DNAzyme, which cleaves the elongated fragment at the RNA site, releasing the probe for repetitive cycling and signal amplification. The cleaved fragment is detected by a reporter molecular beacon. Enzymatic amplification with rapid turnover allows detection of telomerase in the range of 0.1 to 1 μg cell lysate, with a fivefold increase in signal level for cancer cells over normal cells. This probe design can provide a simple, yet rapid and sensitive, measurement of telomerase activity.     

Amplified electrochemical DNA sensor using peroxidase-like DNAzyme

A novel electrochemical DNA sensor was developed here by using peroxidase-like G-quadruplex-based DNAzyme as a biocatalytic label. A hairpin structure including the G-quadruplex-based DNAzyme in a caged configuration and the target DNA probe were immobilized on Au-electrode surface. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated on the electrode surface, triggering the electrochemical oxidization of hydroquinone by H₂O₂, which provide a quantitative measure for the detection of the target DNA. The DNA target was analyzed with a detection limit of 0.6 nM. This method is simple and easy to design without direct conjugation of redox-active element.     

Enzyme‐Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells

Bioorthogonal control of metal‐ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18‐base pair recognition site of a homing endonuclease (I‐SceI), which is found by chance only once in 7×10¹⁰ bp of genomic sequences, and can thus form a near bioorthogonal pair with I‐SceI for DNAzyme activation with minimal effect on living cells. Once I‐SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg²⁺ to release the fluorophore‐labeled DNA fragment and produce a fluorescent turn‐on signal for Mg²⁺. Thus I‐SceI bioorthogonally activates the 10–23 DNAzyme for imaging of Mg²⁺ in HeLa cells.     

A ligation-triggered DNAzyme cascade for amplified fluorescence detection of biological small molecules with zero-background signal

Many types of fluorescent sensing systems have been reported for biological small molecules. Particularly, several methods have been developed for the recognition of ATP or NAD(+), but they only show moderate sensitivity, and they cannot discriminate either ATP or NAD(+) from their respective analogues. We have addressed these limitations and report here a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based amplified detection to develop a ligation-triggered DNAzyme cascade, resulting in ultrahigh sensitivity. First, the 8-17 DNAzyme is split into two separate oligonucleotide fragments as the building blocks for the DNA ligation reaction, thereby providing a zero-background signal to improve overall sensitivity. Next, a CAMB strategy is further employed for amplified signal detection achieved through cycling and regenerating the DNAzyme to realize the true enzymatic multiple turnover (one enzyme catalyzes the cleavage of several substrates) of catalytic beacons. This combination of zero-background signal and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of 100 and 50 pM for ATP and NAD(+), respectively, much lower than those of previously reported biosensors. Moreover, by taking advantage of the highly specific biomolecule-dependence of the DNA ligation reaction, the developed DNAzyme cascades show significantly high selectivity toward the target cofactor (ATP or NAD(+)), and the target biological small molecule can be distinguished from its analogues. Therefore, as a new and universal platform for the design of DNA ligation reaction-based sensing systems, this novel ligation-triggered DNAzyme cascade method may find a broad spectrum of applications in both environmental and biomedical fields.     

Design of a High-Sensitivity Dimeric G-Quadruplex/Hemin DNAzyme Biosensor for Norovirus Detection

G-quadruplexes can bind with hemin to form peroxidase-like DNAzymes that are widely used in the design of biosensors. However, the catalytic activity of G-quadruplex/hemin DNAzyme is relatively low compared with natural peroxidase, which hampers its sensitivity and, thus, its application in the detection of nucleic acids. In this study, we developed a high-sensitivity biosensor targeting norovirus nucleic acids through rationally introducing a dimeric G-quadruplex structure into the DNAzyme. In this strategy, two separate molecular beacons each having a G-quadruplex-forming sequence embedded in the stem structure are brought together through hybridization with a target DNA strand, and thus forms a three-way junction architecture and allows a dimeric G-quadruplex to form, which, upon binding with hemin, has a synergistic enhancement of catalytic activities. This provides a high-sensitivity colorimetric readout by the catalyzing H₂O₂-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline -6-sulfonic acid) diammonium salt (ABTS). Up to 10 nM of target DNA can be detected through colorimetric observation with the naked eye using our strategy. Hence, our approach provides a non-amplifying, non-labeling, simple-operating, cost-effective colorimetric biosensing method for target nucleic acids, such as norovirus-conserved sequence detection, and highlights the further implication of higher-order multimerized G-quadruplex structures in the design of high-sensitivity biosensors.     

Signal Propagation in Multi‐Layer DNAzyme Cascades Using Structured Chimeric Substrates

Signal propagation through enzyme cascades is a critical component of information processing in cellular systems. Although such systems have potential as biomolecular computing tools, rational design of synthetic protein networks remains infeasible. DNA strands with catalytic activity (DNAzymes) are an attractive alternative, enabling rational cascade design through predictable base‐pair hybridization principles. Multi‐layered DNAzyme signaling and logic cascades are now reported. Signaling between DNAzymes was achieved using a structured chimeric substrate (SCS) that releases a downstream activator after cleavage by an upstream DNAzyme. The SCS can be activated by various upstream DNAzymes, can be coupled to DNA strand‐displacement devices, and is highly resistant to interference from background DNA. This work enables the rational design of synthetic DNAzyme regulatory networks, with potential applications in biomolecular computing, biodetection, and autonomous theranostics.     

Nucleic Acid Driven DNA Machineries Synthesizing Mg2+‐Dependent DNAzymes: An Interplay between DNA Sensing and Logic‐Gate Operations

Polymerase/nicking enzymes and nucleic‐acid scaffolds are implemented as DNA machines for the development of amplified DNA‐detection schemes, and for the design of logic gates. The analyte nucleic acid target acts, also, as input for the logic gates. In the presence of two DNA targets, acting as inputs, and appropriate DNA scaffolds, the polymerase‐induced replication of the scaffolds, followed by the nicking of the replication products, are activated, leading to the autonomous synthesis of the Mg²⁺‐dependent DNAzyme or the Mg²⁺‐dependent DNAzyme subunits. These biocatalysts cleave a fluorophore/quencher‐functionalized nucleic‐acid substrate, thus providing fluorescence signals for the sensing events or outputs for the logic gates. The systems are used to develop OR, AND, and Controlled‐AND gates, and the DNA‐analyte targets represent two nucleic acid sequences of the smallpox viral genome.     

A Smart Deoxyribozyme-Programmable Catalytic DNA Circuit for High-Contrast MicroRNA Imaging

Synthetic catalytic DNA circuits have been recognized as a promising signal amplification toolbox for sensitive intracellular imaging, yet their selectivity and efficiency are always constrained by uncontrolled off-site signal leakage and inefficient on-site circuitry activation. Thus, the endogenously controllable on-site exposure/activation of DNA circuits is highly desirable for achieving the selective imaging of live cells. Herein, an endogenously activated DNAzyme strategy was facilely integrated with a catalytic DNA circuit for guiding the selective and efficient microRNA imaging in vivo. To prevent the off-site activation, the circuitry constitute was initially caged without sensing functions, which could be selectively liberated by DNAzyme amplifier to guarantee the high-contrast microRNA imaging in target cells. This intelligent on-site modulation strategy can tremendously expand these molecularly engineered circuits in biological systems.     

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.     

A pH-regulated stimuli-responsive strategy for RNA-cleaving DNAzyme

RNA-cleaving DNAzymes possess important roles in DNAzymes and have been widely used in the biosensors,DNA nanomachines owing to their ion-specific dependence.However,there are still challenges in constructing universal but versatile stimuli-responsive strategies of RNA-cleaving DNAzymes.Herein,a stimuli-responsive strategy for RNA-cleaving DNAzyme is proposed by the artful design of hairpin nanostructure,in which the activities of DNAzyme(Pb~(2+) -dependent DNAzyme as a model) in the hairpin's loop are p H-regulated by using the triplex stem as the "lock".Upon introducing the "key",p H values,the DNAzyme will be activated and fragment the substrate of it in the presence of Pb~(2+),accompanied by the turn-on of the fluorescence quenched by fluorescence resonance energy transfer(FRET).The regulation ability of p H can be controlled by the length and sequence of the triplex stem,and the wide p H regulation range may be helpful for the application of DNAzymes in biological medicine delivery systems.     

Target‐stimulated DNAzyme Concatamers Released from Aptasensor for Highly Sensitive and Specific Detection of Progesterone

Aiming at the detection of ultralow concentration target progesterone (Pro), a novel electrochemical aptasensor based on DNAzyme concatamers signal amplification strategy was proposed. The strategy consists of target DNA strands (TDNAs), and two different hairpin DNA molecules (H1 and H2). The signal is amplified by the large amount of DNAzyme. The TDNAs modified on the electrode open H1 structures in sequence and propagate a reaction of hybridization events between two alternating hairpins (H1and H2) to obtain abundant DNAzyme concatamers. Upon target Pro introduction, a specific Pro‐TDNAs reaction was executed, thereby resulting in the release of DNAzyme concatamers from the electrode. Subsequent differential pulse voltammetry(DPV) detection of aminoazobenzene (DAP) resulting by DNAzyme catalyze the oxidation of o‐phenylenediamine (OPD) with the aid of hydrogen peroxide (H₂O₂). Likewise, a small amount of target Pro can efficiently induce the release of a large number of the DNAzyme from the electrode in the form of DNAzyme concatamer. Under optimal conditions, the the proposed assay presents good electrochemical responses for determination of target Pro in the range of 0.5 to 15 ng/mL with the detection limit of 0.36 ng/mL. In addition, the resulting sensor can successfully distinguish Pro from coexisting interfering substance and show good stability and high repeatability. What's more, the methodology has also been demonstrated by assaying Pro‐spiked samples in serum.     

Enzyme-free signal amplification in the DNAzyme sensor via target-catalyzed hairpin assembly

A simple, highly sensitive and enzyme-free DNAzyme sensor based on target-catalyzed hairpin assembly is developed, which permits detection of 0.1 pM target DNA. Furthermore, this DNAzyme sensor is capable of detecting target DNA in real samples because of its high selectivity.     

Label-free visual detection of nucleic acids in biological samples with single-base mismatch detection capability

We have combined an allosteric molecular beacon for target recognition and guanine-rich DNAzyme for signal amplification to develop a new platform for visual detection of nucleic acids with single-base mismatch detection capability. The fully DNA-structured platform can undergo color change in response to target DNA/RNA, which enables sensitive and selective visual detection in biological samples.     

Ligase-based ultrasensitive detection of DNAzyme cleavage product using molecular beacon

Detection for deoxyribozyme(DNAzyme) cleavage usually needs complex and time-consuming radial labeling,gel electrophoresis and autoradiography.A new approach was reported for detection DNAzyme cleavage product based on molecular beacon (MB).Part of the loop of MB was designed to complementary to DNAzyme cleavage product.MB was employed to monitor ligation process of RNA/DNA complex and to convert directly cleavage product information into fluorescence signal.Detection limit of the assay is 0.02 nmol/L.The cleavage product of 8 -17 DNAzyme against HCV-RNA was detected perfectly based on this assay.The method is fast,simple and ultrasensitive,which might hold great promise in DNAzyme reaction and DNAzyme gene therapy.     

Signal-amplification detection of small molecules by use of Mg2+- dependent DNAzyme

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg²⁺-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg²⁺-dependent DNAzyme is activated, facilitating hybridization between the Mg²⁺-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg²⁺ ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol?L^?1 and detect ATP specifically with a detection limit as low as 13 nmol?L^?1. Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.     

A Thermophilic Tetramolecular G‐Quadruplex/Hemin DNAzyme

The quadruplex‐based DNAzyme system is one of the most useful artificial enzymes or catalysts; their unique properties make them reliable alternatives to proteins for performing catalytic transformation. The first prototype of a thermally stable DNAzyme system is presented. This thermophilic DNAzyme is capable of oxidizing substrates at high temperatures (up to 95 °C) and long reaction times (up to 18 h at 75 °C). The catalytic activity of the DNAzymes were investigated with the standard peroxidase‐mimicking oxidation of 2,2′‐azino‐bis(3‐ethylbenzothiozoline‐6‐sulfonic acid) (ABTS) by H₂O₂. The step‐by‐step design of this unique heat‐activated G‐quadruplex/hemin catalyst, including the modification of adenines at both ends of G‐tracts, the choice of cation, and its concentration for DNAzyme stabilization, is described. This work investigates thoroughly the molecular basis of these catalytic properties and provides an example of an industrially relevant application.     

Amplified nucleic acid sensing using programmed self-cleaving DNAzyme

A newly designed target-assisted self-cleavage (TASC) probe composed of a target-binding site and a DNAzyme domain undergoes TASC when activated via hybridization with a target DNA/RNA. This self-splicing or self-dissociation reaction occurs in a catalytic manner with the probe as a substrate and the target as a catalyst, since the fragmented products are automatically released from the target, thus amplifying the sequence information of the latter under non-PCR, i.e., isothermal and enzyme/reagent-free, conditions. A fluorescence-reporting TASC probe having a fluorescein/dabsyl FRET pair across the cleavage site allows a mix-and-read discrimination of single-nucleotide differences in the target.     

Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells

DNAzymes, which are sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. Despite their significant promise, cellular sensing using DNAzymes has however been difficult, mainly because of the “always‐on” mode of first‐generation DNAzyme sensors. To overcome this limitation, a photoactivatable (or photocaged) DNAzyme was designed and synthesized, and its application in sensing Zn^II in living cells was demonstrated. In this design, the adenosine ribonucleotide at the scissile position of the 8–17 DNAzyme was replaced by 2′‐O‐nitrobenzyl adenosine, rendering the DNAzyme inactive and thus allowing its delivery into cells intact, protected from nonspecific degradation within cells. Irradiation at 365 nm restored DNAzyme activity, thus allowing the temporal control over the sensing activity of the DNAzyme for metal ions. The same strategy was also applied to the GR‐5 DNAzyme for the detection of Pb^II, thus demonstrating the possible scope of the method.     

Accelerated color change of gold nanoparticles assembled by DNAzymes for simple and fast colorimetric Pb2+ detection

The combination of high metal selectivity of DNAzymes with the strong distance-dependent optical properties of metallic nanoparticles has presented considerable opportunities for designing colorimetric sensors for metal ions. We previously communicated a design for a colorimetric lead sensor based on the assembly of gold nanoparticles by a Pb(2+)-dependent DNAzyme. However, heating to 50 degrees C followed by a cooling process of approximately 2 h was required to observe the color change. Herein we report a new improved design that allows fast (<10 min) detection of Pb(2+) at ambient temperature. This improvement of sensor performance is a result of detailed studies of the DNAzyme and nanoparticles, which identified "tail-to-tail" nanoparticle alignment, and large (42 nm diameter) nanoparticle size as the major determining factors in allowing fast color changes. The optimal conditions for other factors such as temperature (35 degrees C) and concentrations of the DNAzyme (2 microM), its substrate (3 nM), and NaCl (300 mM) have also been determined. These results demonstrate that fundamental understanding of the DNAzyme biochemistry and nanoparticle science can lead to dramatically improved colorimetric sensors.