An Exo III-powered closed-loop DNA circuit architecture for biosensing/imaging.

With their regulated Boolean logic operations in vitro and in vivo, DNA logic circuits have shown great promise for target recognition and disease diagnosis. However, significant obstacles must be overcome to improve their operational efficiency and broaden their range of applications. In this study, we propose an Exo III-powered closed-loop DNA circuit (ECDC) architecture that integrates four highly efficient AND logic gates. The ECDC utilizes Exo III as the sole enzyme-activated actuator, simplifying the circuit design and ensuring optimal performance. Moreover, the use of Exo III enables a self-feedback (autocatalytic) mechanism in the dynamic switching between AND logic gates within this circulating logic circuit. After validating the signal flow and examining the impact of each AND logic gate on the regulation of the circuit, we demonstrate the intelligent determination of miR-21 using the carefully designed ECDC architecture in vitro. The proposed ECDC exhibits a linear detection range for miR-21 from 0 to 300 nM, with a limit of detection (LOD) of approximately 0.01 nM, surpassing most reported methods. It also shows excellent selectivity for miR-21 detection and holds potential for identifying and imaging live cancer cells. This study presents a practical and efficient strategy for monitoring various nucleic acid-based biomarkers in vitro and in vivo through specific sequence modifications, offering significant potential for early cancer diagnosis, bioanalysis, and prognostic clinical applications.

pH-Controlled Resettable Modular DNA Strand-Displacement Circuits.

Sophisticated dynamic molecular systems with diverse functions have been fabricated by using the fundamental tool of toehold-mediated strand displacement (TMSD) in the field of dynamic DNA nanotechnology. However, simple approaches to reset these TMSD-based dynamic systems are lacking due to the difficulty in creating kinetically favored pathways to implement the backward resetting reactions. Here, we develop a facile proton-driven strategy to achieve complete resetting of a modular DNA circuit by integrating a pH-responsive intermolecular CG-C+ triplex DNA and an i-motif DNA into the conventional DNA substrate. The pH-programmed strategy allows modular DNA components to specifically associate/dissociate to promote the forward/backward TMSD reactions, thereby enabling the modular DNA circuit to be repeatedly operated at a constant temperature without generating any DNA waste products. Leveraging this tractable approach, we further constructed two resettable DNA logic gates used for logical computation and two resettable catalytic DNA systems with good performance in signal transduction and amplification.

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.

An Aptamer-Functionalized DNA Circuit to Establish an Artificial Interaction between T Cells and Cancer Cells.

Nongenetic strategies that enable control over the cell-cell interaction network would be highly desired, particularly in T cell-based cancer immunotherapy. In this work, we developed an aptamer-functionalized DNA circuit to modulate the interaction between T cells and cancer cells. This DNA circuit was composed of recognition-then-triggering and aggregation-then-activation modules. Upon recognizing target cancer cells, the triggering strand was released to induce aggregation of immune receptors on the T cell surface, leading to an enhancement of T cell activity for effective cancer eradication. Our results demonstrated the feasibility of this DNA circuit for promoting target cancer cell-directed stimulation of T cells, which, consequently, enhanced their killing effect on cancer cells. This DNA circuit, as a modular strategy to modulate intercellular interactions, could lead to a new paradigm for the development of nongenetic T cell-based immunotherapy.

A test strip electrochemical disposable by 3D MXA/AuNPs DNA-circuit for the detection of miRNAs.

The simple and reliable detection of microRNAs is of great significance for studying the biological functions, molecular diagnosis, disease treatment and targeted drug therapy of microRNA. In this study, we introduced a novel Ti3C2Tx (MXene) aerogels (denoted as MXA) composite gold nano-particles (AuNPs)-modified disposable carbon fiber paper (CFP) electrode for the label-free and sensitive detection of miRNA-155. Firstly, in the presence of MXene, graphene oxide (GO) and ethylenediamine (EDA), the 3D MXene hydrogel was formed by self-assembly method, and then adding the freeze-dried 3D MXA dropwise to CFP. Subsequently, electrodepositing AuNPs on the CFP/MXA was done to construct a 3D disposable DNA-circuit test strip with excellent interface. Under the optimum experimental conditions, the detection limit of 3D disposable DNA circuit strip for miRNA-155 was 136 aM (S/N = 3). The CFP/MXA/AuNPs (CMA) electrode also has a wide dynamic range (20 fM to 0.4 µM), with a span of 4 orders of magnitude. Notably, we also tested the practicality of the sensor in 8 clinical samples. The technological innovations in the detection and quantification of microRNA in this work may be helpful to the study new aspects of microRNA biology and the development of diagnosis.

On-Site Non-enzymatic Orthogonal Activation of a Catalytic DNA Circuit for Self-Reinforced In Vivo MicroRNA Imaging.

The wide extracellular-intracellular distribution of microRNA requires the on-site, robust and efficient activation of catalytic DNA circuits inside live cells. Herein, we develop an efficient non-enzymatic circuitry activation strategy to realize the orthogonally controlled catalytic DNA (CCD) circuit for achieving high-fidelity in vivo microRNA imaging through multiply guaranteed molecular recognition and progressively accelerated signal amplification. For predictable on-site activation and useful catalytic efficiency, the dominating circuitry fuel strand was initially split into inactive fuel subunits that were grafted into an auxiliary catalytic circuit. There, the in-cell-specific mRNA triggered the orthogonal amplification of the active fuel strands for sensitive target detection through the chief entropy-driven catalytic DNA circuit. We believe that the on-site orthogonal circuitry activation method can contribute to clinical diagnosis and prognosis.

Construction of an Autocatalytic Hybridization Assembly Circuit for Amplified In Vivo MicroRNA Imaging.

Lowly expressed analyte in complex cytoplasmic milieu necessitates the development of non-enzymatic autocatalytic DNA circuits with high amplification and anti-interference performance. Herein, we engineered a versatile and robust stimuli-responsive autocatalytic hybridization assembly (AHA) circuit for high-performance in vivo bioanalysis. Under a moderately confined condition, the initiator motivated the autonomous and cooperative cross-activation of cascade hybridization reaction and catalytic DNA assembly for generating an exponentially amplified readout without the parasite steric hindrance and random diffusion side effects. The AHA circuit was systematically investigated by a series of experimental studies and theoretical simulations. The successively guaranteed target recognition and synergistically accelerated signal-amplification enabled the sensitive and selective detection of analyte, and realized the robust miRNA imaging in living cells and mice. This autocatalytic DNA circuit could substantially expand the toolbox for accurate diagnosis and programmable therapeutics.

A Chimeric Conjugate of Antibody and Programmable DNA Nanoassembly Smartly Activates T Cells for Precise Cancer Cell Targeting.

Synthetically directing T-cells against tumors emerges as a promising strategy in immunotherapy, while it remains challenging to smartly engage T cells with tunable immune response. Herein, we report an intelligent molecular platform to engineer T-cell recognition for selective activation to potently kill cancer cells. To this end, we fabricated a hybrid conjugate that uses a click-type DNA-protein conjugation to equip the T cell-engaging antibody with two distinct programmable DNA nanoassemblies. By integrating multiple aptameric antigen-recognitions within a dynamic DNA circuit, we achieved combinatorial recognition of triple-antigens on cancer cells for selective T-cell activation after high-order logic operation. Moreover, by coupling a DNA nanostructure, we precisely defined the valence of the antigen-binding aptamers to tune avidity, realizing effective tumor elimination in vitro and in vivo. Together, we present a versatile and programmable strategy for synthetic immunotherapy.

Disposable 3D GNAs/AuNPs DNA-Circuit Strip for miRNAs Dynamic Quantification.

Real-time quantitative monitoring of miRNAs plays an essential role in diagnosis and therapeutics. Herein, a DSN-coupled graphene nanoarray/gold nanoparticles (GNAs/AuNPs) carbon paper (CP) electrode for the dynamic, sensitive, and real-time analysis of miRNAs is reported. GNAs are vertically grown on the conductive CP by radio frequency plasma enhanced chemical vapor deposition, and AuNPs are electrodeposited on CP/GNAs to build a 3D ultrasensitive sensing interface with large specific surface area, good conductivity and biocompatibility. The dynamic quantitative monitoring of microRNA-21 (miR-21) is realized by cyclic voltammetry with a series of different concentrations within 16 min, and this 3D GNAs/AuNPs DNA-circuit strip shows good performance for the simultaneous detection of miR-21 and miR-155, and the detection limits are as low as 21.4 and 30.3 am, respectively. Moreover, comparable detection results are achieved for clinical samples between the proposed sensor and qRT-PCR, suggesting the reliability of the constructed sensor. This ultrasensitive sensing and disposable DNA-circuit strip with 3D structure can efficiently shorten the diffusion distance between reactive biomolecules and the sensing interface, enhance the hybridization of probes and improve the sensitivity of the biosensor, holding great promise for the rapid, quantitative and dynamic monitoring of multiple low concentrations of biomolecules in point-of-care clinical analysis.

Colorimetric nanoplatform for visual determination of cancer cells via target-catalyzed hairpin assembly actuated aggregation of gold nanoparticles.

According to aptamer-mediated hairpin DNA cascade amplifier and gold nanoparticles aggregation, an optical platform for cancer cells determination has been proposed. High-affinity chimeric aptamers were used for cancer cell detection and also as an initiator for beginning hairpin assembly to construct three-way junction (3WJ) nanostructures. These three hairpins were modified at 3' ends with biotin. In the presence of target cell, chimeric aptamer binds to its ligand on cell surface and initiates 3WJ nanostructures formation. These 3WJ nanostructures interact with streptavidin-modified gold nanoparticles (AuNPs) via non-covalent biotin-streptavidin interactions and create a crossover lattice of nanoparticles. This event leads to AuNPs aggregation and red-shifting. The results were confirmed by gel electrophoresis and UV-visible spectrophotometry. The dynamic range of this assay is 25 to 107 cells with a detection limit of 10 cells which is respectively 9 and 4 times more significant than the sensitivity of AuNP-based approaches without amplification and enzyme-mediated signal amplification. Graphical abstract.

Dynamically Programmed Switchable DNA Hydrogels Based on a DNA Circuit Mechanism.

Biological stimuli-responsive DNA hydrogels have attracted much attention in the field of medical engineering owing to their unique phase transitions from gel to sol through cleavage of DNA cross-linking points in response to specific biomolecular inputs. In this paper, a new class of biological stimuli-responsive DNA hydrogels with a dynamically programmed DNA system that relies on a DNA circuit system through cascading toehold-mediated DNA displacement reactions is constructed, allowing the catalytic cleavage of cross-linking points and main chains in response to an appropriate DNA input. The dynamically programmed DNA hydrogels exhibit a significant sharp phase transition from gel to sol in comparison to another DNA hydrogel showing noncatalytic cleavage of cross-linking points due to synchronization of the catalytic cleavage of cross-linking points and the main chains. Further, the sol-gel phase transitions of the DNA hydrogels in response to the DNA input are easily tunable by changing the cross-linking density. Additionally, with a structure-switching aptamer, DNA hydrogels encapsulating PEGylated gold nanoparticles can be used as enzyme-free signal amplifiers for the colorimetric detection of adenosine 5'-triphosphate (ATP); this detection system provides simplicity and higher sensitivity (limit of detection: 5.6 × 10-6 m at 30 min) compared to other DNA hydrogel-based ATP detection systems.

An Efficient Particle-Based DNA Circuit System: Catalytic Disassembly of DNA/PEG-Modified Gold Nanoparticle-Magnetic Bead Composites for Colorimetric Detection of miRNA.

An efficient particle-based DNA circuit system for a new colorimetric miRNA assay is designed and devised based on a catalytic disassembly strategy through a target miRNA-triggered DNA circuit mechanism. The new particle-based DNA circuit system shows a rapid color change as well as significant improvement of sensitivity without need for other enzymes or instruments.

Inhibition of kinetic random-distribution in DNA Seesaw gates and biosensors for complete leakage prevention.

DNA nanomaterials have a wide application prospect in biomedical field, among which DNA computers and biosensors based on Seesaw-based DNA circuit is considered to have the most development potential. However, the serious leakage of Seesaw-based DNA circuit prevented its further development and application. Moreover, the existing methods to suppress leakage can't achieve the ideal effect. Interestingly, we found a new source of leakage in Seesaw-based DNA circuit, which we think is the main reason why the previous methods to suppress leakage are not satisfactory. Therefore, based on this discovery, we use DNA triplex to design a new method to suppress the leakage of Seesaw-based DNA circuit. Its ingenious design makes it possible to perfectly suppress the leakage of all sources in Seesaw-based DNA circuit and ensure the normal output of the circuit. Based on this technology, we have constructed basic Seesaw module, AND gate, OR gate, secondary complex circuits and DNA detector. Experimental results show that we can increase the working range of the secondary Seesaw-based DNA circuit by five folds and keep its normal output signal above 90%, and we can improve the LOD of the Seesaw-based DNA detector to 1/11 of the traditional one(1.8pM). More importantly, we successfully developed a detector with adjustable detection range, which can theoretically achieve accurate detection in any concentration range. We believe the established triplex blocking strategy will greatly facilitate the most powerful Seesaw based DNA computers and biosensors, and further promote its application in biological systems.

Scaling up of a Self-Confined Catalytic Hybridization Circuit for Robust microRNA Imaging.

The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self-assembly system. Herein, a catalytic self-defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre-sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self-assembly via a microRNA (miRNA)-powered self-sorting catalytic hybridization reaction. With minimal strand complexity, this self-sustainable CSC system streamlined the circuit component and achieved localization-intensified cascaded signal amplification. Profiting from the self-adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para-carcinoma tissues. The CSC-sustained self-assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment.

An electrochemical biosensor designed with entropy-driven autocatalytic DNA circuits for sensitive detection of ovarian cancer-derived exosomes.

Intelligent artificial DNA circuits have emerged as a promising approach for modulating signaling pathways and signal transduction through rational design, which may contribute to comprehensively realizing biomolecular sensing of organisms. In this work, we have fabricated an electrochemical biosensor for the sensitive and accurate detection of ovarian cancer-derived exosomes by constructing an entropy-driven autocatalytic DNA circuit (EADC). Specifically, the robust EADC is prepared by the self-assembly of well-designed DNA probes, and upon stimulation of the presence of ovarian cancer cells-derived exosomes, numerous inputs can be produced to feedback and accelerate the reaction. The catalytic abilities of the generated input sequences play a pivotal role in EADC and dramatically enhance the signal amplification capability. Through the combination of the autocatalytic circuit and circular cleavage reactions, significantly changed electrochemical signals can be recorded for sensitive analysis of the exosomes with a remarkably low detection limit of 30 particles/µL. Moreover, the proposed enzyme-free biosensor shows exceptional performance in distinguishing patient samples from healthy samples, which exhibits promising prospects for the clinical diagnosis of ovarian cancer.

An enzymatically activated AND-gate DNA logic circuit for tumor cells recognition via multi-microRNAs detection.

The DNA-based logic circuit, constructed to mimic biochemical reaction networks, is highly significant in detecting biomarkers at the molecular level. The differences in the expression levels of microRNAs (miRNAs) within different types of cells provide hope for distinguishing cell subtypes. However, reliance on a single miRNA often leads to unreliable results. Herein, we constructed an enzyme-triggered cascade logic circuit based on the AND gate, which is capable of generating corresponding fluorescence signals in the presence of target miRNAs. The introduction of apurinic/apyrimidinic (AP) sites effectively reduces the likelihood of false signal generation. Amplification of the fluorescence signal relies on the catalytic hairpin assembly and the repetitive reuse of the multicomponent nucleic acid enzyme (MNAzyme). We demonstrated that the logic circuit can not only distinguish cancer cells from normal cells but also identify different types of cancer cells. The programmability of the logic circuits and the simplicity of the assay system allow us to modify the functional sequences to recognize different types of biomarkers, thus providing a reference for the identification of various cell subtypes.

Programmable Primer Switching for Regulating Enzymatic DNA Circuits.

Developing DNA strand displacement reactions (SDRs) offers crucial technical support for regulating artificial nucleic acid circuits and networks. More recently, enzymatic SDR-based DNA circuits have gained significant attention because of their modular design, high orthogonality signaling, and extremely fast reaction rates. Typical enzymatic SDRs are regulated by relatively long primers (20-30 nucleotides) that hybridize to form stable double-stranded structures, facilitating enzyme-initiated events. Implementing more flexible primer-based enzymatic SDR regulations remains challenging due to the lack of convenient and simple primer control mechanism, which consequently limits the development of enzymatic DNA circuits. In this study, we propose an approach, termed primer switching regulation, that implements programmable and flexible regulations of enzymatic circuits by introducing switchable wires into the enzymatic circuits. We applied this method to generate diverse enzymatic DNA circuits, including cascading, fan-in/fan-out, dual-rail, feed-forward, and feedback functions. Through this method, complex circuit functions can be implemented by just introducing additional switching wires without reconstructing the basic circuit frameworks. The method is experimentally demonstrated to provide flexible and programmable regulations to control enzymatic DNA circuits and has future applications in DNA computing, biosensing, and DNA storage.

Protein-to-DNA Converter with High Signal Gain.

DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

An approach for state differentiation in nucleic acid circuits: Application to diagnostic DNA computing.

BACKGROUND: Differentiating between different states in nucleic acid circuits is crucial for various biological applications. One approach, there is a requirement for complicated sequential summation, which can be excessive for practical purposes. By selectively labeling biologically significant states, this study tackles the issue and presents a more cost-effective and streamlined solution. The challenge is to efficiently distinguish between different states in a nucleic acid circuit. RESULTS: An innovative method is introduced in this study to distinguish between states in a nucleic acid circuit, emphasizing the biologically relevant ones. The circuit comprises four DNA logic gates and two detection modules, one for determining fetal gender and the other for diagnosing X-linked genetic disorders. The primary module generates a G-quadruplex DNAzyme when activated by specific biomarkers, which leads to a distinct colorimetric signal. The secondary module responds to hemophilia and choroideremia biomarkers, generating one or two DNAzymes. The absence of female fetus indicators results in no DNAzyme or color change. The circuit can differentiate various fetal states by producing one to four active DNAzymes in response to male fetus biomarkers. A single-color solution for state differentiation is provided by this approach, which promises significant advancements in DNA computing and diagnostic applications. SIGNIFICANCE: The innovative approach used in this study to distinguish states in nucleic acid circuits holds great significance. By selectively labeling biologically relevant states, circuit design is simplified and complexity is reduced. This advancement enables cost-effective and efficient diagnostic applications and contributes to DNA computing, providing a valuable solution to a fundamental problem.

Latent Toehold-Mediated DNA Circuits Based on a Bulge-Loop Structure for Leakage Reduction and Its Application to Signal-Amplifying DNA Logic Gates.

DNA circuits based on successive toehold-mediated DNA displacement reactions, particularly entropy-driven DNA circuit (EDC) systems, have attracted considerable attention as powerful enzyme-free tools for dynamic DNA nanotechnology. However, background leakage (noise signal) often occurs when the circuit is executed nonspecifically, even in the absence of the appropriate catalyst DNA (input). This study designed and developed a new latent toehold-mediated DNA circuit (LDC) system that relies on a bulge-loop structure as a latent toehold toward leakage reduction. Furthermore, the number (size) of nucleotides (nt) in the bulge-loop is found to play a significant role in the performance (i.e., leakage, signal, and kinetics) of LDC systems. In fact, the signal rate for the LDC systems increased as the number of nt in the bulge-loop increased from 4 to 8, whereas the leakage rate of the LDC systems with bulge-loops of 7 nt or less was low, but the leakage rate of the LDC system with a bulge-loop of 8 nt increased significantly. Note that the LDC system with the optimal bulge-loop (7 nt) was capable of not only reducing the leakage but also accelerating the circuit speed without any signal loss, unlike methods of reducing the leakage by reducing the signal reported previously for the conventional EDC systems. These facts indicate that the 7 nt bulge-loop acts as a "latent" toehold for the DNA circuit system. By using the amplification function of output signals with an accelerated circuit and reduced leakage, our LDC system with a 7 nt bulge-loop could be applied directly and successfully to signal-amplifying DNA logic gates such as OR and AND gates, and thus, sufficient output signals could be obtained even with a small amount of input. These findings reveal that our LDC systems with a bulge-loop structure can replace the conventional EDC system and have enormous potential in the field of DNA nanotechnology.