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G-quadruplex-based complexes have been widely used in various analytical methods due to their outstanding capabilities of generating colorimetric, fluorescent or electrochemical signals. However, since loop sequences in traditional G-quadruplex structures are quite short, it is difficult to establish biosensors solely using G-quadruplex-based complexes. Herein, we attempted to lengthen the loop sequences of G-quadruplex structures and found that G-quadruplex-hemin DNAzymes (G-DNAzymes) with long loops (even 30 nucleotides) maintain high peroxidase activity. In addition, the peroxidase activity is not affected by the hybridization of the long loop with its complementary counterpart. Consequently, G-DNAzyme can be endowed with an additional function by taking the long loop as a recognition element, which may facilitate the construction of diverse colorimetric biosensors. Furthermore, by designing an apurinic/apyrimidinic site or a complementary sequence of microRNA-21 (miRNA-21) in long loops, bifunctional G-DNAzymes can be split in the presence of apurinic/apyrimidinic endonuclease 1 (APE1) or miRNA-21, decreasing their peroxidase activities. Accordingly, APE1 and miRNA-21 are quantified using 3,3',5,5'-tetramethylbenzidine as a chromophore. Using the G-DNAzyme, APE1 can be detected in a linear range from 2.5 to 22.5 U mL -1 with a LOD of 1.8 U mL -1 . It is to be noted that benefitting from duplex-specific nuclease-induced signal amplification, the linear range of the miRNA-21 biosensor is broadened to 5 orders of magnitude, while the limit of detection is as low as 73 fM. This work demonstrates that G-DNAzymes with long loops can both generate signals and recognize targets, providing an alternative strategy to design G-quadruplex-based analytical methods.

Synthetic molecular robots can execute sophisticated molecular tasks at nanometer resolution. However, a molecular robot capable of controlling cellular behavior remains unexplored. Herein, we report a self-propelled DNA robot operating on the cell membrane to control the migration of a cell. Driven by DNAzyme catalytic activity, the DNA robot could autonomously and stepwise move on the membrane-floating cell-surface receptors in a stochastic manner and simultaneously trigger the receptor-dimerization to activate downstream signaling for cell motility. The cell membrane-associated continuous motion and operation of a DNA robot allowed for the ultrasensitive regulation of MET/AKT signaling and cytoskeleton remodeling to enhance cell migration. Finally, we designed distinct conditional DNA robots to orthogonally manipulate the cell migration in a coculture of mixed cell populations. We have developed a novel strategy to engineer a cell-driving molecular robot, representing a promising avenue for precise cell manipulation with nanoscale resolution., (© 2021 Wiley-VCH GmbH.)

In this work, a multiply-amplified peroxidase-like colorimetric strategy was proposed for the high-specific recognition and ultrasensitive detection of kanamycin (Kana). Based on two Kana-aptamer triggered sequential reactions, G-quadruplex (G4) and DNA (hairpins) modified Ni-Fe layered double oxides (LDOs) could be obtained simultaneously. Later, a three-dimensional G4/LDO frame networks, as a novel DNAzyme, with enhanced peroxidase-like catalytic activity was assembled through electrostatic interaction. This DNAzyme catalyzed 3,3',5,5'-tetramethylbenzidine oxidation for the colorimetric detection of Kana. The enhancement principle was discussed and the charge transfer process during the catalytic reaction was investigated. Under the optimal experiment conditions, the proposed method exhibited high sensitivity, where the linear range is from 10 fM to 10 nM (r 2  = 0.992), and the limit of detection is 3 fM (S/N = 3). The practicability of this assay was demonstrated by successfully application of residual Kana detection in genuine milk and urine samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Numerical computation is a focus of DNA computing, and matrix operations are among the most basic and frequently used operations in numerical computation. As an important computing tool, matrix operations are often used to deal with intensive computing tasks. During calculation, the speed and accuracy of matrix operations directly affect the performance of the entire computing system. Therefore, it is important to find a way to perform matrix calculations that can ensure the speed of calculations and improve the accuracy. This paper proposes a DNA matrix operation method based on the mechanism of the DNAzyme binding to auxiliary strands to cleave the substrate. In this mechanism, the DNAzyme binding substrate requires the connection of two auxiliary strands. Without any of the two auxiliary strands, the DNAzyme does not cleave the substrate. Based on this mechanism, the multiplication operation of two matrices is realized; the two types of auxiliary strands are used as elements of the two matrices, to participate in the operation, and then are combined with the DNAzyme to cut the substrate and output the result of the matrix operation. This research provides a new method of matrix operations and provides ideas for more complex computing systems.

RNA-cleaving DNAzymes and their multicomponent nucleic acid enzymes (MNAzymes) have been successfully used to detect nucleic acids and proteins. The appropriate split of the catalytic cores of DNAzymes is critical to the formation of MNAzymes with high catalytic activities. However, for protein detection, no systematic investigation has been made on the effects of the split locations and secondary structures of MNAzymes on the catalytic activities of the cleavage reaction. We systematically studied how split locations and secondary structures affect the activity of the MNAzymes that catalyze multiple cleavage steps. We engineered the MNAzymes on the basis of the RNA-cleaving DNAzyme 10-23 as a model system. We designed 28 pairs of MNAzymes, representing 14 different split locations and two secondary structures: the three-arm and the four-arm structures. By comparing the multiple turnover numbers ( k obs.m ) of the 28 MNAzymes, we showed that the split location between the seventh cytosine and the eighth thymine of the catalytic core region and the four-arm structure resulted in optimum catalytic activity. Binding-induced DNA assembly of the optimized MNAzymes enabled sensitive detection of two model protein targets, demonstrating promising potential of the binding-assembled MNAzymes for protein analysis. The strategy of binding-assembled MNAzymes and systematic studies measuring multiple turnover numbers ( k obs.m ) provide a new approach to studying other partial (split) DNAzymes and engineering better MNAzymes for the detection of specific proteins.

Herein, a photocurrent polarity switching platform for highly selective assay of mucin 1 (MUC1) was developed based on target-induced hemin transfer from ZrO 2 hollow spheres (ZrO 2 HSs) to G-quadruplex nanowires (G wires). In this system, SiO 2 spheres were used as templates to synthesize the uniform and mesoporous ZrO 2 HSs. As nanocontainers, ZrO 2 HSs could load hemin in its cavity via pores. Then, the aptamers of MUC1, as bio-gates, blocked the pores of ZrO 2 HSs based on the specific binding of Zr 4+ and the phosphate groups of aptamer. In the presence of MUC1, the aptamer could specifically recognize and bind with MUC1, and then leave away from the surface of ZrO 2 HSs, which resulted in the opening of the bio-gates and releasing of hemin. Assisted with the G wires formed on the Au NPs/In 2 S 3 /ITO, the released hemin was captured on the electrode through the formation of hemin/G-quadruplex structure, leading to the switch of the photocurrent polarity of the electrode from anodic photocurrent to cathodic photocurrent. The proposed photoelectrochemical biosensor showed outstanding performance for MUC1 assay with high selectivity, wide linear response range (1 fg mL -1 -10 ng mL -1 ) and lower detection limit (0.48 fg mL -1 ). And the strategy could be easily extended to a triple-mode detection of MUC1 because the hemin/G-quadruplex structure was widely used in electrochemical and colorimetric methods as a hydrogen peroxide mimetic enzyme, which might provide wide applications in biological or clinical studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A novel and low-cost DNAzyme, Ni/Fe layered double hydroxide (LDH) nanosheet/G-quadruplex (without hemin) with enhanced peroxidase-mimic activity was designed. The catalytic mechanism was investigated. The detection of Cu(II) in actual serum samples could be realized sensitively via this efficient DNAzyme-based method.

The development of multifunctional nanoplatforms that integrate both diagnostic and therapeutic functions has always been extremely desirable and challenging in the cancer combat. Here, we report an endogenous miRNA-activated DNA nanomachine (EMDN) in living cells for concurrent sensitive miRNA imaging and activatable gene silencing. EMDN is constructed by interval hybridization of two functional DNA monomers (R/HP and F) to a DNA nanowire generated by hybridization chain reaction. After the target cell-specific transportation of EMDN, intracellular let-7a miRNA initiates the DNA nanomachine by DNA strand displacement cascades, resulting in an amplified fluorescence resonance energy-transfer signal and the release of many free HP sequences. The restoration of HP hairpin structures further activates the split-DNAzyme to identify and cleave the EGR-1 mRNA to realize gene silencing therapy. The proposed EMDN shows efficient cell internalization, good biological stability, rapid reaction kinetics, and the ability to avoid false-positive signals, thus ensuring reliable miRNA imaging in living cells. Meanwhile, the controlled activation of the split-DNAzyme activity regulated by the intracellular specific miRNA may be promising in the precise treatment of cancer. Collectively, this strategy provides a valuable nanoplatform for early clinical diagnosis and activatable gene therapy of tumors.

Transmembrane signal transduction is of profound significance in many biological processes. The dimerization of cell-surface receptors is one prominent mechanism by which signals are transmitted across the membrane and trigger intracellular cascade amplification reactions. Recreating such processes in artificial systems has potential applications in sensing, drug delivery, bioengineering, and providing a new route for a deep understanding of fundamental biological processes. However, it remains a challenge to design artificial signal transduction systems working by the receptor dimerization mechanism in a predictable and smart manner. Here, benefitting from DNA with features of programmability, controllability, and flexible design, we use DNA as a building material to construct an artificial system mimicking dimerization of receptors for signal transduction and cascade amplification. DNA-based membrane-spanning receptor analogues are designed to recognize external signals, which drives two receptors into close spatial proximity to activate DNAzymes inside the cell-mimicking system. The activation of the DNAzyme initiates the catalyzed cleavage of encapsulated substrates and leads to the release of fluorescent second messengers for signal amplification. Such an artificial signal transduction system extends the range of biomimetic DNA-based signaling systems, providing a new avenue to study natural cell signaling processes and artificially regulate biological processes.

Listeria monocytogenes is an important foodborne pathogen, which imposes great burdens on public health. The current methods for detecting L. monocytogene are limited in several ways such as time consuming and lab equipment dependent. In this study, we developed a new electrochemical assay to improve the efficacy. This assay allows us to generate numerous G-quadruplex sequences while loop-mediated isothermal amplification happens. Then, these G-quadruplex sequences form DNAzyme to produce a color change and an electrochemical signal by oxidizing tetramethylbenzidine. This assay could be finished in 2 h, which significantly reduced the detection time. Also, we confirmed the limit of detection of this assay at 6.8 CFU/mL according to 3σ criterion. Our assay shows good sensitivity to detect bacteria range from 52.5 to 5.25 × 10 4 CFU/mL. This assay's reliability was also confirmed by detecting artificially contaminated pork samples. Thus, we propose this electrochemical assay for rapid and sensitive detection of L. monocytogenes in food., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site-specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Adenosine 5'-triphosphate (ATP) is a central extracellular signaling agent involved in various physiological and pathological processes. However, precise measurements of the temporal and spatial components of ATP dynamics are lacking due primarily to the limitations of available methods for ATP detection. Here, we report on the first effort to design a self-phosphorylating DNAzyme (SPDz) sensor for fluorescence imaging of ATP. In response to ATP, SPDz sensors exhibit subsecond response kinetics, extremely high specificity, and micromolar affinities. In particular, we demonstrate cell-surface-anchored SPDz sensors for fluorescence imaging of both stress-induced endogenous ATP release in astrocytes and mechanical stimulation-evoked ATP release at the single-cell level. We also validated their utility for visualizing the rapid dynamic properties of ATP signaling upon electrical stimulation in astrocytes. Thus, SPDz sensors are robust tools for monitoring ATP signaling underlying diverse cellular processes.

Pathogens have long presented a significant threat to human lives, and hence the rapid detection of infectious pathogens is vital for improving human health. Current detection methods lack the means to detect infectious pathogens in a simple, rapid, and reliable manner at the time and point of need. Functional nucleic acids (FNAs) have the potential to overcome these limitations by acting as key components for point-of-care (POC) biosensors due to their distinctive advantages that include high binding affinities and specificities, excellent chemical stability, ease of synthesis and modification, and compatibility with a variety of signal-amplification and signal-transduction mechanisms.This Account summarizes the work completed in our groups toward developing FNA-based biosensors for detecting bacteria. In vitro selection has led to the isolation of many RNA-cleaving fluorogenic DNAzymes (RFDs) and DNA aptamers that can recognize infectious pathogens, including Escherichia coli , Clostridium difficile , Helicobacter pylori , and Legionella pneumophila. In most cases, a "many-against-many" approach was employed using a DNA library against a crude cellular mixture of an infectious pathogen containing diverse biomarkers as the target to isolate RFDs, with combined counter and positive selections ensuring high specificity toward the desired target. This procedure allows for the isolation of pathogen-specific FNAs without first identifying a suitable biomarker. Multiple target-specific DNA aptamers, including anti-glutamate dehydrogenase (GDH) circular aptamers, anti-degraded toxin B aptamers, and anti-RNase HII aptamers, have also been isolated for the detection of bacteria such as Clostridium difficile . The isolated FNAs have been integrated into fluorescent, colorimetric, and electrochemical biosensors using various signal transduction mechanisms. Both simple-to-use paper-based analytical devices and hand-held electrical devices with integrated FNAs have been developed for POC applications. In addition, signal-amplification strategies, including DNA catenane enabled rolling circle amplification (RCA), DNAzyme feedback RCA, and an all-DNA amplification system using a four-way junction and catalytic hairpin assembly (CHA), have been designed and applied to these systems to further increase their detection sensitivity. The use of these FNA-based biosensors to detect pathogens directly in clinical samples, such as urine, blood, and stool, has now been demonstrated with an outstanding sensitivity of as low as 10 cells per milliliter, highlighting the tremendous potential of using FNA-based sensors in clinical applications. We further describe strategies to overcome the challenges of using FNA-based biosensors in clinical applications, including strategies to improve the stability of FNAs in biological samples and prevent their nonspecific degradation from nucleases and strategies to deal with issues such as signal loss caused by nonspecific binding and biofouling. Finally, the remaining roadblocks for employing FNA-based biosensors in clinical applications are discussed.

In 8-17 DNAzyme, the end loop A 6 G 7 C 8 is a highly conserved motif. Here we reported an activation approach by specific chemical modifications on A6 and C8 for more efficient Ca 2+ -mediated reaction. The importance of the end loop was further highlighted and its critical conservation broken for more powerful catalysts., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Molecular probe that enables in vivo imaging is the cornerstone of accurate disease diagnosis, prognostic estimation, and therapies. Although several nucleic acid-based probes have been reported for tumor detection, it is still a challenge to develop programmable methodology for accurately identifying tumors in vivo . Herein, a reconfigurable DNA hybridization-based reaction was constructed to assemble DNAzyme computing that contains an intracellular miRNA-unlocked entropy-driven catalysis module and an endogenous metal ion-responsive DNAzyme module for specific in vivo imaging. By reasonable design, the programmable DNAzyme computing can not only successfully distinguish tumor cells from normal cells but also enable tumor imaging in living mice. Due to its excellent operation with high specificity and sensitivity, this design may be broadly applied in the biological study and personalized medicine.

The sensitive assays for protein, especially for the DNA-based assay, are often dependent on the amplification procedure with assistance of enzyme. Compared with a protein enzyme, deoxyribozyme (DNAzyme) exhibits similar catalytic activity and specificity and better flexibility. In this work, we streamlined the binding induced DNA displacement (BINDD) strategy for the activation of DNAzyme cleavage. Since the intrinsic element of DNAzyme is the nucleic acids, it is easy to join the BINDD by hybridization with an affinity probe. The activity of DNAzyme was initiated by the BINDD reaction mediated by the recognition affinity probe with target proteins. Upon DNAzyme release, it was able to catalyze and cleave the predesigned substrates, generating the enhanced fluorescence signal indicating the protein concentration. Such a constructed homogeneous assay is available and effective in human serum when it was used for detection of platelet derived growth factor-BB (PDGF-BB) and prostate specific antigen (PSA), with detection limits of 100 pM and 200 pM, respectively., (© 2021 John Wiley & Sons, Ltd.)

Direct measurement of DNA repair is critical for the annotation of their clinical relevance and the discovery of drugs for cancer therapy. Here we reported a "repaired and activated" DNAzyme (RADzyme) by incorporating a single methyl lesion (O 6 MeG, 3MeC, or 1MeA) at designated positions through systematic screening. We found that the catalytic activity of the RADzyme was remarkably suppressed and could be restored via enzyme-mediated DNA repair. Benefit from these findings, a fluorogenic RADzyme sensor was developed for the monitoring of MGMT-mediated repair of O 6 MeG lesion. Importantly, the sensor allowed the evaluation of MGMT repair activity in different cells and under drugs treatment. Furthermore, another RADzyme sensor was engineered for the monitoring of ALKBH2-mediated repair of 3MeC lesion. This strategy provides a simple and versatile tool for the study of the basic biology of DNA repair, clinical diagnosis and therapeutic assessment., (© 2021 Wiley-VCH GmbH.)

During its development in recent decades, DNAzyme has become a promising candidate for application in biosensor field. However, it still suffers from the problem of thermodynamic and biological instability such as nuclease digestion, which limits its applications in complex samples. Here we have presented a simple and common strategy to resolve this problem by engineering the linear DNAzyme into a circular shape DNAzyme based on the integration of substrate and enzyme parts into one single-stranded sequence. This circular DNAzyme system is indeed endowed with excellent stability due to the stable intramolecular double-stranded formation and extraordinary resistance to nuclease digestion due to the closed structure. We demonstrated that this circular DNAzyme system gained excellent stability and could active under conditions across a broader range of temperature, salt concentrations, and pH. Depending on this circular DNAzyme, combing with Terminal deoxynucleotidyl transferase (TdT)-generated G-quadruplexes, a label free colorimetric sensing platform for Pb 2+ quantitation was developed, and a detection limit of 0.085 nM was achieved. Then the enzyme digestion cycle amplification was introduced to further improve the sensitivity of the sensing system, an ultralow detection limit of 0.0015 nM for this fluorescence method was achieved. Based on the two sensing platforms, ultrasensitive analysis of Pb 2+ in environmental water and food samples was successfully realized. It is anticipated that this stable circular DNAzyme design will be helpful for trace detection in complex samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This article provides a comprehensive review of biosensing with DNAzymes, providing an overview of different sensing applications while highlighting major progress and seminal contributions to the field of portable biosensor devices and point-of-care diagnostics. Specifically, the field of functional nucleic acids is introduced, with a specific focus on DNAzymes. The incorporation of DNAzymes into bioassays is then described, followed by a detailed overview of recent advances in the development of in vivo sensing platforms and portable sensors incorporating DNAzymes for molecular recognition. Finally, a critical perspective on the field, and a summary of where DNAzyme-based devices may make the biggest impact are provided.

Abnormal levels of reduced glutathione (GSH) and glutathione reductase (GR) are usually related to a variety of diseases, so it is of great significance to determine the GSH concentration and GR activity. We herein develop a smartphone-assisted colorimetric biosensor for the detection of GSH and GR activity in human serum and mouse liver using hemin/G-quadruplex DNAzyme. Firstly, an obvious color change from colorless to green can be observed, owing to the high peroxidase-like activity of hemin/G-quadruplex DNAzyme toward 2,2'-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS). With the addition of GSH or GR, the H 2 O 2 -mediated oxidation of ABTS catalyzed by hemin/G-quadruplex DNAzyme is significantly inhibited, resulting in remarkable color fading. Therefore, the detection of GSH and GR activity can be achieved by observing the color transition or measuring the absorbance at 420 nm. The detection limit was estimated to be as low as 0.1 μM and 10 μU/mL for GSH and GR, respectively. More interestingly, the RGB values of the sensing system can be identified by the smartphone application (APP, color collect), which makes it an ideal format for on-site determination and point-of-care testing (POCT). In addition, the proposed method shows excellent selectivity and acceptable applicability for the determination of GSH concentration and GR activity in human serum samples and mouse liver tissues, which might hold great application potential in clinical diagnosis and drug screening.

The conditional control of protein function in response to the physiological change of cells is of great interest for studying protein function in biological settings and developing protein therapeutics. We report herein that catalase (CAT) DNAzyme can potentiate the generation of reactive oxygen species (ROS) in living cells by knocking down catalase expression, which could further activate a reactive oxygen species (ROS)-responsive pro-protein, RNase A-NBC, in situ. Using an optimized lipid nanoparticle delivery system to simultaneously introduce CAT DNAzyme and RNase A-NBC into cells, we show that the pro-protein, RNase A-NBC, could be activated in a significantly enhanced manner to prohibit tumor cell growth in different types of cancer cells. We believe the methodology of regulating pro-protein activity using DNAzyme biocatalysis to differentiate intracellular environment could further be extended to other functional proteins, and even fundamental investigations in living systems to develop pro-protein therapeutics., (© 2021 Wiley-VCH GmbH.)

Functional DNA nanostructures have been widely used in various bioassay fields. Yet, the programmable assembly of functional DNA nanostructures in living cells still represents a challenging goal for guaranteeing the sensitive and specific biosensing utility. In this work, we report a self-catalytic DNA assembly (SDA) machine by using a feedback deoxyribozyme (DNAzyme)-amplified branched DNA assembly. This SDA system consists of catalytic self-assembly (CSA) and DNAzyme amplification modules for recognizing and amplifying the target analyte. The analyte initiates the CSA reaction, leading to the formation of Y-shaped DNA that carries two RNA-cleaving DNAzymes. One DNAzyme can then successively cleave the corresponding substrate and generate numerous additional inputs to activate new CSA reactions, thus realizing a self-catalytic amplification reaction. Simultaneously, the other DNAzyme is assembled as a versatile signal transducer for cleaving the fluorophore/quencher-modified substrate, leading to the generation of an amplified fluorescence readout. By incorporating a flexible auxiliary sensing module, the SDA system can be converted into a universal sensing platform for detecting cancerous biomarkers, e.g., a well-known oncogene microRNA-21 (miR-21). Moreover, the SDA system realized the precise intracellular miR-21 imaging in living cells, which is attributed to the reciprocal amplification property between CSA reactions and DNAzyme biocatalysis. This compact SDA amplifier machine provides a universal and facile toolbox for the highly efficient identification of cancerous biomarkers and thus holds great potential for early cancer diagnosis.

DNAzymes have emerged as an important class of sensors for a wide variety of metal ions, with florescence DNAzyme sensors as the most widely used in different sensing and imaging applications because of their fast response time, high signal intensity, and high sensitivity. However, the requirements of an external excitation light source and its associated power increase the cost and size of the fluorometer, making it difficult to be used for portable detections. To overcome these limitations, we report herein a DNAzyme sensor that relies on chemiluminescence resonance energy transfer (CRET) without the need for external light. The sensor is constructed by combining the functional motifs from both Pb 2+ -dependent 8-17 DNAzyme conjugated to fluorescein (FAM) and hemin/G-quadruplex that mimics horseradish peroxidase to catalyze the oxidation of luminol by H 2 O 2 to yield chemiluminescence. In the absence of Pb 2+ , the hybridization between the enzyme and substrate strands bring the FAM and hemin/G-quadruplex in close proximity, resulting in CRET. The presence of Pb 2+ ions can drive the cleavage on the substrate strand, resulting in a sharp decrease in the melting temperature of hybridization and thus separation of the FAM from hemin/G-quadruplex. The liberated CRET pair causes a ratiometric increase in the donor's fluorescent signal and a decrease in the acceptor signal. Using this method, Pb 2+ ions have been measured rapidly (<15 min) with a low limit of detection at 5 nM. By removing the requirement of exogenous light excitation, we have demonstrated a simple and portable detection using a smartphone, making the DNAzyme-CRET system suitable for field tests of lake water. Since DNAzymes selective for other metal ions or targets, such as bacteria, can be obtained using in vitro selection, the method reported here opens a new avenue for rapid, portable, and ratiometric detection of many targets in environmental monitoring, food safety, and medical diagnostics.

Extracellular ATP (eATP) is an important biological signal transduction molecule. Although a variety of detection methods have been extensively used in ATP sensing and analysis, accurate detection of eATP remains difficult due to its extremely low concentration and spatiotemporal distribution. Here, an eATP measurement strategy based on tetrahedral DNA (T-DNA)-modified electrode sensing platform and hybridization chain reaction (HCR) combined with G-quadruplex/Hemin (G4/Hemin) DNAzyme dual signal amplification is proposed. In this strategy, ATP aptamer and RNA-cleaving DNAzyme were combined to form a split aptazyme. In the presence of ATP, this aptazyme hydrolyzes the cleaving substrate strand with high selectivity, releasing cleaved ssDNA, which are captured by the T-DNA assembled on the electrode surface, triggering an HCR on the electrode surface to form numerous linker sequences of the HCR dsDNA product. When G-quadruplex@AuNPs (G4) spherical nucleic acid enzymes (SNAzymes) with other linkers are used as nanocatalyst tags, they are captured by HCR dsDNA through sticky linkers present on the electrode surface. An amplified electrochemical redox current signal is generated through SNAzyme-mediated catalysis of H 2 O 2 , enabling easy detection of picomole amounts of ATP. Using this strategy, eATP levels released by tobacco suspension cells were accurately measured and the distribution and concentration of eATP released on the surface of an Arabidopsis leaf was determined., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The catalytic cycle of a peroxidase-mimicking heme-DNAzyme involves an iron(IV)oxo porphyrin π-cation radical intermediate known as compound I formed through heterolytic O-O bond cleavage of an Fe 3+ -bound hydroperoxo ligand (Fe-OOH) in compound 0 , like that of a heme enzyme such as horseradish peroxidase (HRP). Peroxidase assaying of complexes composed of chemically modified hemes possessing various electron densities of the heme iron atom (ρ Fe ) and parallel-stranded tetrameric G-quadruplex DNAs of oligonucleotides d(TTAGGG), d(TTAGGGT), and d(TTAGGGA) was performed to elucidate the effects of the heme electronic structure and local heme environment on the catalytic activity of the heme-DNAzyme. The study revealed that the DNAzyme activity is enhanced through an increase in the ρ Fe and general base catalysis of the adenine base adjacent to the heme, which are reminiscent of the "push" and "pull" mechanisms in the catalytic cycle of HRP, respectively, and that the activity of the heme-DNAzyme can be independently controlled through the heme electronic structure and local heme environment. These findings allow a deeper understanding of the structure-function relationship of the peroxidase-mimicking heme-DNAzyme.

Testing multiple biomarkers, as opposed to one, has become a preferred approach for diagnosing many heterogeneous diseases, such as cancer and infectious diseases. However, numerous technologies, including gold standard ELISA and PCR, can detect only one type of biomarker, either protein or nucleic acid (NA), respectively. In this work, we report for the first time simultaneous detection of proteins and NAs in the same solution, using solely functional NA (FNA) molecules. In particular, we combined the thrombin binding aptamer (TBA) and the 10-23 RNA-cleaving DNA enzyme (DNAzyme) in a single aptazyme molecule (Aptazyme 1.15-3' ), followed by extensive optimization of buffer composition, sequences and component ratios, to establish a competitive bioassay. Subsequently, to establish a multiplex bioassay, we designed a new aptazyme (Aptazyme 2.20-5' ) by replacing the target recognition and substrate sequences within Aptazyme 1.15-3' . This designing process included an in silico study, revealing the impact of the target recognition sequence on the aptazyme secondary structure and its catalytic activity. After proving the functionality of the new aptazyme in a singleplex bioassay, we demonstrated the capability of the two aptazymes to simultaneously detect thrombin and NA target, or two NA targets in a multiplex bioassay. High specificity in target detection was achieved with the limits of detection in the low nanomolar range, comparable to the singleplex bioassays. The presented results deepen the barely explored features of FNA for diagnosing multiple targets of different origins, adding an extra functionality to their catalogue., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K + or NH 4 + ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.

Sensitive detection of the SARS-CoV-2 protein remains a great research interest in clinical screening and diagnosis owing to the coronavirus epidemic. Here, an ultrasensitive chemiluminescence (CL) imaging strategy was developed through proximity hybridization to trigger the formation of a rolling circle-amplified G-quadruplex/hemin DNAzyme for the detection of the SARS-CoV-2 protein. The target protein was first recognized by a pair of DNA-antibody conjugates, Ab-1 and Ab-2, to form a proximity-ligated complex, Ab-1/SARS-CoV-2/Ab-2, which contained a DNA sequence complemental to block DNA and thus induced a strand displacement reaction to release the primer from a block/primer complex. The released primer then triggered a rolling circle amplification to form abundant DNAzyme units in the presence of hemin, which produced a strong chemiluminescent signal for the detection of the target protein by catalyzing the oxidation of luminol by hydrogen peroxide. The proposed assay showed a detectable concentration range over 5 orders of magnitude with the detection limit down to 6.46 fg/mL. The excellent selectivity, simple procedure, acceptable accuracy, and intrinsic high throughput of the imaging technique for analysis of serum samples demonstrated the potential applicability of the proposed detection method in clinical screening and diagnosis.

Functional nucleic acids (FNAs), including naturally occurring ribozymes and riboswitches as well as artificially created DNAzymes and aptamers, have been popular molecular toolboxes for diverse applications. Given the high chemical stability of nucleic acids and their ability to fold into diverse sequence-dependent structures, FNAs are suggested to be highly functional under unusual reaction conditions. This review will examine the progress of research on FNAs under conditions of low pH, high temperature, freezing conditions, and the inclusion of organic solvents and denaturants that are known to disrupt nucleic acid structures. The FNA species to be discussed include ribozymes, riboswitches, G-quadruplex-based peroxidase mimicking DNAzymes, RNA-cleaving DNAzymes, and aptamers. Research within this space has not only revealed the hidden talents of FNAs but has also laid important groundwork for pursuing these intriguing functional macromolecules for unique applications., (© 2021 Wiley-VCH GmbH.)

In this study, we have designed a three-fluorophore-labeled Y-shaped DNAzyme with a high catalytic cleavage activity and a three-dimensional (3D) MOF-MoS 2 NB (metal-organic framework fused with molybdenum disulfide nanobox), which was synthesized as an efficient quencher of the fluorescent biosensor. The synthesized porous 3D MOF-MoS 2 NBs and Y-shaped DNAzyme exhibited a good analytical response toward the simultaneous multiple detections of Hg 2+ , Ni 2+ , and Ag + ions over the other coexisting metal ions. More specifically, the three kinds of enzyme aptamer and substrate aptamer (SA) were hybridized and annealed to form the Y-shaped DNAzyme structure and labeled with three different fluorophores such as FAM, TAMRA, and ROX over the 3'-end of SA. When the targets were induced, the DNAzyme was triggered to cleave the fluorophore-labeled SAs. Then, the cleaved SAs (FAM-SA, TAMRA-SA, and ROX-SA) were adsorbed on the 3D MOF-MoS 2 NB surface to quench the fluorescence signal due to a noncovalent interaction (van der Waals and π-π stacking interaction), which transmuted the fluorescence on-state to off-state. As a result, the fluorescence assay confiscated the high selectivity and sensitivity for the target analytes of Hg 2+ , Ni 2+ , and Ag + ions achieved for the detection limits of 0.11 nM, 7.8 μM, and 0.25 nM, respectively. Accordingly, the sensitivity of the developed sensor was explored with a better lower detection limit than the previously reported biosensors. The utility of the designed Y-shaped DNAzyme may find a broad field of application in real water sample analysis with interfering contaminants.

Artificial evolution experiments typically use libraries of ∼1015 sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. To test this idea, we investigated the catalytic potential of DNA architectures in which twelve or fifteen randomized positions were embedded in a scaffold present in all library members. After incubating in either the presence or absence of lead (which promotes the nonenzymatic cleavage of RNA), library members that cleaved themselves at an RNA linkage were purified by PAGE and characterized by high-throughput sequencing. These selections yielded deoxyribozymes with activities 8- to 30-fold lower than those previously isolated under similar conditions from libraries containing 1014 different sequences, indicating that the disadvantage of using a less diverse pool can be surprisingly small. It was also possible to elucidate the sequence requirements and secondary structures of deoxyribozymes without performing additional experiments. Due to its relative simplicity, we anticipate that this approach will accelerate the discovery of new catalytic DNA and RNA motifs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Trihydroxyphenyl porphyrin (POH3) was designed to specifically bind with a triplex DNA resulting in a turn-on fluorescence response. This ensemble can be developed into a catalytic triplex DNAzyme towards porphyrin metalation. The catalytic activity is initiated by the enhanced basicity of POH3 upon binding with the triplex DNA.

The RNA-cleaving 8-17 DNAzyme, which is a metalloenzyme that depends on divalent metal ions for its function, is the most studied catalytic DNA in terms of its mechanism. By the end of 2017, a report of the crystal structure of the enzyme-substrate complex in the presence of Pb 2+ probed some of the previous findings and opened new questions, especially around the participation of the metal ion in the catalytic mechanism and the promiscuity exhibited by the enzyme in terms of the metal cofactor required for catalysis. In this article we explore the role of the divalent metal ion in the mechanism of the 8-17 DNAzyme as a general acid, by measuring the influence of pH over the activity of a slower variant of the enzyme in the presence of Pb 2+ . We replaced G14, which has been identified as a general base in the mechanism of the enzyme, by the unnatural analog 2-aminopurine, with a lower pK a value of the N1 group. With this approach, we obtained a bell-shaped pH-rate profile with experimental pK a values of 5.4 and 7.0. Comparing these results with previous pH-rate profiles in the presence of Mg 2+ , our findings suggest the stabilization of the 5'-O leaving group by the hydrated metal ion acting as a general acid, in addition to the activation of the 2'-OH nucleophile by the general base G14.

The RNA-cleaving 17E DNAzyme exhibits different levels of cleavage activity in the presence of various divalent metal ions, with Pb 2+ giving the fastest cleavage. In this study, the metal-phosphate interaction is probed to understand the trend of activity with different metal ions. For the first-row transition metals, the lowest activity shown by Ni 2+ correlates with the inhibition by the inorganic phosphate and its water ligand exchange rate, suggesting inner-sphere metal coordination. Cleavage activity with the two stereoisomers of the phosphorothioate-modified substrates, R p and S p , indicated that Mg 2+ , Mn 2+ , Fe 2+ , and Co 2+ had the highest S p : R p activity ratio of >900. Comparatively, the activity was much less affected using the thiophilic metals, including Pb 2+ , suggesting inner-sphere coordination. The pH-rate profiles showed that Pb 2+ was different than the rest of the metal ions in having a smaller slope and a similar fitted apparent p K a and the p K a of metal-bound water. Combining previous reports and our current results, we propose that Pb 2+ most likely plays the role of a general acid while the other metal ions are Lewis acid catalysts interacting with the scissile phosphate.

Despite the great efforts of using DNAzyme for gene therapy, its clinical success is limited by the lack of simple delivery systems and limited anticancer efficacy. Here, we develop a simple approach for the synthesis of hybrid nanostructures that exclusively consist of DNAzyme and Cu 2+ with ultra-high loading capacity. The Cu-DNAzyme nanohybrids allow to effectively co-deliver DNAzyme and Cu 2+ into cancer cells for combinational catalytic therapy. The released Cu 2+ can be reduced to Cu + by glutathione and then catalyze endogenous H 2 O 2 to form cytotoxic hydroxyl radicals for chemodynamic therapy (CDT), while the 10-23 DNAzyme enables the catalytic cleavage of VEGFR2 mRNA and activates gene silencing for gene therapy. We demonstrate that the system can efficiently accumulate in the tumor and exhibit amplified cascade antitumor effects with negligible systemic toxicity. Our work paves an extremely simple way to integrate DNAzyme with CDT for the dual-catalytic tumor treatment., (© 2021 Wiley-VCH GmbH.)

DNA-hydrolyzing DNAs represent an attractive type of DNA-processing catalysts distinctive from the protein-based restriction enzymes. The innate DNA property has enabled them to readily join DNA-based manipulations to promote the development of DNA biotechnology. A major in vitro selection strategy to identify these DNA catalysts relies tightly on the isolation of linear DNAs processed from a circular single-stranded (ss) DNA sequence library by self-hydrolysis. Herein, we report that by programming a terminal hybridization stem in the library, other than the previously reported classes (I & II) of deoxyribozymes, two new classes (III & IV) were identified with the old selection strategy to site-specifically hydrolyze DNA in the presence of Zn2+. Their representatives own a catalytic core consisting of ∼20 conserved nucleotides and a half-life of ∼15 min at neutral pH. In a bimolecular construct, class III exhibits unique broad generality on the enzyme strand, which can be potentially harnessed to engineer DNA-responsive DNA hydrolyzers for detection of any target ssDNA sequence. Besides the new findings, this work should also provide an improved approach to select for DNA-hydrolyzing deoxyribozymes that use various molecules and ions as cofactors., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

As bisphenol A (BPA) is harmful to human health, it is of great significance to develop a new method for BPA detection. Herein, we designed a BPA biosensor by integrating an amplifying DNA circuit with Mg 2+ -dependent DNAzyme into the sensing system. The BPA-aptamer binding activated a DNA circuit for signal amplification based on toehold-mediated strand displacement. A catalytic Mg 2+ -dependent DNAzyme was formed through synergistically DNA hybridization, which can cleave the dual-labeled substrate DNA into two segments. The separation of the fluorophore and quencher produces a high fluorescence response for BPA detection. This biosensor exhibited a superior sensitivity with a detection limit of 50 fM. The method is selective and robust, which can work even in milk samples with satisfactory accuracy. The biosensor analytical results were also verified by liquid chromatography coupled with mass spectrometry (LC-MS) and no obvious difference existed between the two methods., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Several circulating miRNAs are associated with the pathogenic process of acute myocardial infarction (AMI). Thus, analyzing myocardial miRNAs in the circulatory system is important for the diagnosis and treatment of AMI, especially for early-stage diagnosis. Based on the characteristics of myocardial miRNAs, an ultrasensitive and multitargeted electrochemiluminescence (ECL) sensing platform was developed with a versatile probe that can couple DNAzyme with hybridization chain reaction amplification. The target miRNA and auxiliary chains form a circular unit that shears the versatile probe hairpin, and the products subsequently trigger cascading amplification; a long strand of dsDNA is then generated with many C-rich sequences that can undergo in situ reductions to generate ECL luminophore silver clusters. Using this strategy, three myocardial miRNAs are successfully detected with a detection limit as low as 29.6 aM ( S / N = 3). Notably, our method can detect myocardial miRNA groups composed of multiple related circulating miRNAs with high selectivity over interfering miRNAs in blood. This is extremely important for solving the problem of diverse and low abundance of infarct-associated miRNAs. Our strategy pioneers a new idea of miRNA detection, and given its versatility and sensitivity, it is promising for the diagnosis of multigene-regulated cardiovascular diseases.

Although chemotherapy is one of the most widely used cancer treatments, there are serious side effects, drug resistance, and secondary metastasis. To address these problems, herein we designed a bimetallic metal-organic framework (MOF) encapsulated with DNAzyme for co-triggered in situ cancer drug synthesis and DNAzyme-based gene therapy. Once in cancer cells, MOFs would disassemble and liberate copper ions, zinc ions, and DNAzyme under the acidic environment of lysosomes. Copper ions can catalyze the synthesis of the chemotherapeutic drug through copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction after being reduced to Cu I ; zinc ions act as the cofactor to activate the cleavage activity of DNAzyme. The anticancer drug is synthesized intracellularly and can kill cancer cells on site to minimize side effects to normal organisms. The activated DNAzyme starts gene therapy to inhibit tumor proliferation and metastasis by targeting and cleaving oncogene substrates., (© 2021 Wiley-VCH GmbH.)

Multi-signal synergistically amplified electrochemical sensing of HIV DNA was proposed based on two-dimensional (2D) DNA-Au nanowire structure coupled with hemin/G-quadruplex and polymerase chain reaction (PCR). In the design, by using target HIV DNA as the template, PCR generated numbers of double-stranded DNA (dsDNA) with free single-stranded DNA (ssDNA) tails on one side and free G-quadruplex sequences on the other side. Then, the ssDNA tails of the PCR products were hybridized with the capture probe (CP) to introduce the hemin/G-quadruplex to the electrode surface as a redox-active reporter and to amplify the electrochemical signal as mimic peroxidase catalysis in the presence of H 2 O 2 . Meanwhile, (+)AuNPs were electrostatically adsorbed onto dsDNA surface for the formation of 2D DNA-Au nanowire structure, amplifying the electrochemical signal further as another mimic peroxidase and electric conductor together. By effectively combining these signal amplification processes, ultrasensitive HIV DNA detection was achieved with a detection limit of 1.3 aM, indicating that it has potential application in clinical diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA-based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA-cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation-inspired molecular device for controlling gene expression and cancer treatment., (© 2021 Wiley-VCH GmbH.)

The epigenetic modification of nucleic acids represents a versatile approach for achieving high-efficient control over gene expression and transcription and could dramatically expand their biosensing and therapeutic applications. Demethylase-involved removal of N6-methyladenine (m 6 A) represents one of the vital epigenetic reprogramming events, yet its direct intracellular evaluation and as-guided gene regulation are extremely rare. The endonuclease-mimicking deoxyribozyme (DNAzyme) is a catalytically active DNA that enables the site-specific cleavage of the RNA substrate, and several strategies have imparted the magnificent responsiveness to DNAzyme by using chemical and light stimuli. However, the epigenetic regulation of DNAzyme has remained largely unexplored, leaving a significant gap in responsive DNA nanotechnology. Herein, we reported an epigenetically responsive DNAzyme system through the in vitro selection of an exquisite m 6 A-caged DNAzyme that could be specifically activated by FTO (fat mass and obesity-associated protein) demethylation for precise intracellular imaging-directed gene regulation. Based on a systematic investigation, the active DNAzyme configuration was potently disrupted by the site-specific incorporation of m 6 A modification and subsequently restored into the intact DNAzyme structure via the tunable FTO-specific removal of m 6 A-caging groups under a variety of conditions. This orthogonal demethylase-activated DNAzyme amplifier enables the robust and accurate monitoring of FTO and its inhibitors in live cells. Moreover, the simple demethylase-activated DNAzyme facilitates the assembly of an intelligent self-adaptive gene regulation platform for knocking down demethylase with the ultimate apoptosis of tumor cells. As a straightforward and scarless m 6 A removal strategy, the demethylase-activated DNAzyme system offers a versatile toolbox for programmable gene regulation in synthetic biology.

Potassium ion (K + ) plays a crucial role in biological systems, such as maintaining cellular processes and causing diseases. However, specifically, the detection of K + is extremely challenging because of the coexistence of the chemically similar ion of Na + under physiological conditions. In this work, a K + specific biosensor is constructed on the basis of a dimerized G-quadruplex (GQ) DNA, which is promoted by K + , and the enzymatic activity of the resulting DNAzyme depends on the concentration of the K + . The K + in a 1-200 mM concentration range can be selectively detected by visual color, UV-Vis absorbance or fluorescence even if the concentration of the accompanying Na + is up to 140 mM at an ambient condition up to 45 °C. In addition, this system can also be used to selectively detect NH 4 + in a 5-200 mM concentration range. This dimerized DNAzyme offers a new type of biosensor with a potential application in the biological system.

Preparation of long single-stranded (ss)DNA in large quantities with high efficiency and purity remains a synthetic challenge. Here, we present a protocol for using DNA-hydrolyzing DNA enzymes (deoxyribozymes) for efficient biotechnological production of milligrams of ssDNA with a customizable sequence up to a few kilobases. Our protocol provides a convenient yet economical way to store the sequence information of target ssDNA on phages for selective mass production on demand. For complete details on the use and execution of this protocol, please refer to Jia et al. (2021)., Competing Interests: A China patent (ZLYY239-201032A) on large-scale production of ssDNA nanostructures with DNA-cleaving DNAs has been filed., (© 2021 The Author(s).)

DNAzyme-based gene therapy holds immense prospects for effectively treating severe diseases, yet is constrained with inefficient delivery and unconditional activation. Herein, we designed a bioinspired self-catabolic DNA nanocapsule for sustaining tumor-specific cascade activation of therapeutic DNAzyme. The exquisite DNAzyme was temporarily masked by the self-excising DNAzyme in the hierarchical rolling circle replication (RCR) nanostructures, thus stayed in an inactive state in physiological fluids. Through the multivalent tumor-anchoring aptamer strands, the RCR nanocapsule was specifically accumulated in cancer cells and was sequentially activated for motivating the ultimate DNAzyme-mediated gene silencing via the intelligent stimuli-responsive cascade DNAzyme activation. By virtue of the programmable RCR assembly strategy, our compact DNAzyme nanoplatform shows great promise for developing versatile smart gene therapeutics and personalized nanomedicines., (© 2021 Wiley-VCH GmbH.)

Kras and Braf are major oncogenes. The mutation of Kras codon 12 or Braf V600E can lead to ovarian carcinoma. The detection of oncogene-related DNAs and their mutations offers solution for early diagnosis of ovarian cancer. Herein, a size-tunable multi-functional DNA hexahedral-nanostructure (DHN) has been rationally designed and modified on the electrode to response to Kras and Braf DNA. The size of DHN is controlled via polyadenines (polyA). The complete self-assembly of DHN depends on the presence of both target DNAs and two assistant probes. Meanwhile, a HRP-mimicking DNAzyme forms in DHN, which catalyzes the polymerization of aniline. The produced polyaniline is utilized as the output signal through differential pulse voltammetry (DPV). The biosensor shows the linear range from 100 fM to 1 μM, with the detection limit of 48.7 fM for Kras gene; and the linear range from 100 fM to 100 nM, with the detection limit of 44.1 fM for Braf gene, respectively. Since the current response depends on both gene sequences, the high specificity of the biosensor endows it to operate in an "OR"-type logic gate to discriminate the mutation of both genes. When Kras codon 12 or Braf V600E mutation happens, the response decreases significantly due to the incomplete formation of DNAzyme in DHN. The practicability of the biosensor has been verified through challenging human serum samples. Thus, it has great potential for clinical diagnosis of ovarian cancer through simultaneous detection of Kras and Braf genes and their mutations., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gene silencing holds promise for cancer therapeutics because of its potential to inhibit genes involved in tumor development. However, gene silencing is still restricted by its limited efficacy and safety. Nanoscale coordination polymers (CPs) emerge as promising nanocarriers for gene delivery, but their responsiveness and potential therapeutic properties have rarely been explored simultaneously. Here, multifunctional ultrathin 2D nanosheets of Cu(I) 1,2,4-triazolate CP with a thickness of 4.5 ± 0.8 nm are synthesized using a bottom-up method. These CP nanosheets can act as both an effective DNAzyme nanocarrier for gene therapy and an intrinsic photosensitizer for hypoxia-tolerant type I photodynamic therapy (PDT), which is ascribed to the Fenton-like reaction. Because of the glutathione (GSH)-responsiveness of the CP nanosheets, DNAzyme-loaded CP nanosheets exhibit excellent cancer-cell-targeting gene silencing of the early growth response factor-1 (EGR-1), with messenger RNA inhibited by 84% in MCF-7 (human breast cancer cells) and only 6% in MCF-10A (normal human mammary epithelial cells). After tail intravenous injection into MCF-7-tumor-bearing mice, the CP nanosheets loaded with chlorin-e6-modified DNAzyme under photoirradiation show a high antitumor efficacy (88.0% tumor regression), demonstrating a promising therapeutic platform with efficient and selective gene silencing and PDT of cancer., (© 2021 Wiley-VCH GmbH.)

Regulating cell-cell interactions and cell behaviors via cell surface engineering is of significance for biological research such as cell fate control and cell therapy. While extensive efforts have been made to induce cell-cell assembly via various cell surface modifications triggered by macromolecules or organic metabolites, controllable cell-cell interactions that include both assembly and disassembly triggered by metal ions remain a challenge. Herein, we report a strategy based on DNAzymes to realize controllable cell-cell interactions, triggered by metal ions. The metal-dependent DNAzyme-based cleavage can effectively manipulate cell behaviors, including cell-cell conjunctions and disaggregation. Using a Zn 2+ -specific DNAzyme, a Mg 2+ -specific DNAzyme, and their respective substrate strands as the building blocks, the corresponding DNA double-chain switches enabling two-factor disassembly are demonstrated. Moreover, the method has been applied to control the assembly and disassembly between two cell spheroids. Since a wide variety of metal-specific DNAzymes are available, this method can be readily applied to construct cell dynamic systems controlled by other metal ions, providing a smart and versatile platform to regulate dynamic cell behavior.

The covalent linkage of catalytic units to aptamer sequence-specific nucleic acids exhibiting selective binding affinities for substrates leads to functional scaffolds mimicking native enzymes, nucleoapzymes. The binding of the substrates to the aptamer and their structural orientation with respect to the catalytic units duplicate the functions of the active center of enzymes. The possibility of linking the catalytic sites directly, or through spacer units, to the 5'-end, 3'-end, and middle positions of the aptamers allows the design of nucleoapzyme libraries, revealing structure-functions diversities, and these can be modeled by molecular dynamics simulations. Catalytic sites integrated into nucleoapzymes include DNAzymes, transition metal complexes, and organic ligands. Catalytic transformations driven by nucleoapzymes are exemplified by the oxidation of dopamine or l-arginine, hydroxylation of tyrosine to l-DOPA, hydrolysis of ATP, and cholic acid-modified esters. The covalent linkage of photosensitizers to the tyrosinamide aptamer leads to a photonucleoapzyme scaffold that binds the N -methyl- N '-(3-aminopropane)-4,4'-bipyridinium-functionalized tyrosinamide to the aptamer. By linking the photosensitizer directly, or through a spacer bridge to the 5'-end or 3'-end of the aptamer, we demonstrate a library of supramolecular photosensitizer/electron acceptor photonucleoapzymes mimicking the functions of photosystem I in the photosynthetic apparatus. The photonucleoapzymes catalyze the photoinduced generation of NADPH, in the presence of ferredoxin-NADP + -reductase (FNR), or the photoinduced H 2 evolution catalyzed by Pt nanoparticles. The future prospects of nucleoapzymes and photonucleoapzymes are discussed.