DNA Circuits Research Articles

Regulable toehold lock for the effective control of strand displacement reaction sequence and circuit leakage

Strand displacement reaction enables the construction of enzyme-free DNA reaction networks, thus has been widely applied to DNA circuit and nanotechnology. It has the characteristics of high efficiency, universality and regulatability. However, the existing regulation tools cannot enable effective control of the reaction sequence, which undoubtedly limits the construction of complex nucleic acid circuits. Herein, we developed a regulation tool, toehold lock, and achieved strict control of reaction sequence without loss of the main reaction signal output. Furthermore, we applied the tool to scenarios such as seesaw circuits, AND/OR logic gates, and entropy-driven circuits, and respectively demonstrated its significant superiority compared to the original method. We believe that the proposed toehold lock has greatly optimized the efficiency of DNA strand displacement-based networks, and we anticipate that the tool will be widely used in multiple fields.

Navigable DNA walker coupled with exonucleases-assisted signal amplification for sensitive fluorescent detection of Staphylococcus aureus

A fluorescent aptasensor for Staphylococcus aureus (S. aureus) was constructed by integrating the mechanisms of fluorescence signal regulation and exonucleases-assisted two-step signal amplification into a navigable DNA walker. After the recognition of S. aureus, the first signal amplification step converts the aptamer-cDNA1 duplex to the hydrolysate of cDNA1 by exonuclease I (Exo I). Then the second signal amplification step converts the hydrolysate to the fuel chain of the navigable DNA walker with the help of exonuclease III (Exo III) reaction system containing hairpin probe and cDNA2. Driven by the fuel chain, the movement of the DNA walker triggers the fluorescence resonance energy transfer (FRET) to obtain an amplified fluorescent signal that correlates to the target concentration. The movement track is ingeniously designed and constructed by DNA self-assembly technology, which controls the motion direction and stride of the DNA walker. In addition, the movement triggers FRET, which effectively enhances the detection signal. The biosensor has a wide detection concentration range of 1 × 102-2 × 107 CFU/mL, and the detection limit is 9 CFU/mL. This biosensor provides a reference method for customization of signal amplification strategy in DNA circuit machinery. And has potential applications in the fields of environmental and food safety monitoring.

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

AbstractNongenetic 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 highly sensitive self-powered sensing method designed on DNA circuit strategy and MoS2 hollow nanorods for detection of thalassemia

Thalassemia is one of the most common monogenic diseases, which seriously affects human growth and development, cardiovascular system, liver, etc. There is currently no effective cure for this disease, making screening for thalassemia particularly important. Herein, a self-powered portable device with high sensitivity and specificity for efficiently screening of low-level thalassemia is developed which is enabled with AuNPs/MoS2@C hollow nanorods and triple nucleic acid amplification technologies. It is noteworthy that AuNPs/MoS2@C electrode shows the advantages of high electrocatalytic activity, fast carrier migration rate and large specific surface area, which can significantly improve the stability and output signal of the platform. Using high-efficiency tetrahedral DNA as the probe, the target CD122 gene associated with thalassemia triggers a catalytic hairpin assembly reaction to achieve CD122 recycling while providing binding sites for subsequent hybridization chain reaction, greatly improving the detection accuracy and sensitivity of the device. A reliable electrochemical/colorimetric dual-mode assay for CD122 is then established, with a linear response range of 0.0001–100 pM for target concentration and open circuit voltage, and the detection limit is 78.7 aM (S/N = 3); a linear range of 0.0001–10000 pM for CD122 level and RGB Blue value, with a detection limit as low as 58.5 aM (S/N = 3). This method achieves ultra-sensitive and accurate detection of CD122, providing a new method for the rapid and accurate screening of thalassemia.

Construction of an autocatalysis-driven DNA circuit for highly efficient detection of H5N1 oligonucleotide

H5N1 is a highly pathogenic avian influenza virus (AIV) that is a significant risk to both poultry production and human health. The sensitive and reliable determination of H5N1 is thus highly desirable for early antiviral therapy and controlling the outbreak. Isothermal DNA circuits have afforded a promising tool for molecular sensing in complex biological environments, yet are always constrained by their low amplification depth and insufficient signal gain. Herein, a simple-yet-robust autocatalytic DNA circuit (ADC) was devised for highly efficient H5N1 DNA analysis through the synergistic catalysis of catalytic hairpin assembly (CHA) and rolling circle amplification (RCA). The trigger-activated RCA circuit can generate numerous repeated initiators for stimulating the CHA transducer. Meanwhile, the initiator-stimulated CHA circuit can produce the re-assembled RCA trigger to reversely motivate the RCA system. The synergistic cross-activation between the RCA and CHA amplicon can achieve progressive reaction acceleration and exponentially amplified signal gain. The ADC system is highly versatile and capable of reliably detecting H5N1 DNA in buffer and biological samples with high performance, highlighting its great promise for low-abundance biomolecule sensing and early disease diagnosis.

A visual DNA compilation of Rössler system and its application in color image encryption

This paper proposes the use of visual DNA compilation for the Rössler chaotic system in the context of semi-synthetic biology, which has promising prospects for the development of next-generation computers. The proposed method utilizes DNA toehold mediated strand displacement circuit and fluorescent labeling to construct a DNA circuit as a pseudo-random number generator with high information entropy. To overcome the degeneration of the key space due to the finite precision of concentration detection, the key space is expanded based on the keys of dk. Additionally, concentrations of DNA strand detected in valid time are transformed into diverse multiple blocks to construct chaotic sequences. Experimental results demonstrate that the proposed encryption approach is highly secure and has a strong ability to resist common attacks. Specifically, the proposed image encryption algorithm provides positive security and is resistant to diverse attacks, thus ensuring the information security of the plaintext image.

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.

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

AbstractSynthetic 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.

Programmable Molecular Signal Transmission Architecture and Reactant Regeneration Strategy Driven by EXO λ for DNA Circuits.

The characteristics of DNA hybridization enable molecular computing through strand displacement reactions, facilitating the construction of complex DNA circuits, which is an important way to realize information interaction and processing at a molecular level. However, signal attenuation in the cascade and shunt process hinders the reliability of the calculation results and further expansion of the DNA circuit scale. Here, we demonstrate a novel programmable exonuclease-assisted signal transmission architecture, where DNA strand with toehold employed to inhibit the hydrolysis process of EXO λ is applied in DNA circuits. We construct a series circuit with variable resistance and a parallel circuit with constant current source, ensuring excellent orthogonal properties between input and output sequences while maintaining low leakage (<5%) during the reaction. Additionally, a simple and flexible exonuclease-driven reactant regeneration (EDRR) strategy is proposed and applied to construct parallel circuits with constant voltage sources that could amplify the output signal without extra DNA fuel strands or energy. Furthermore, we demonstrate the effectiveness of the EDRR strategy in reducing signal attenuation during cascade and shunt processes by constructing a four-node DNA circuit. These findings offer a new approach to enhance the reliability of molecular computing systems and expand the scale of DNA circuits in the future.

Efficient Intracellular MicroRNA Imaging Based on the Localized and Self-Sustainable Catalytic DNA Assembly Circuit.

Building dynamic biological networks, especially DNA circuits, has provided a powerful prospect for exploring the intrinsic regulation processes of live cells. Nevertheless, for efficient intracellular microRNA analysis, the available multi-component circuits are constrained by their limited operating speed and efficiency due to the free diffusion of reactants. Herein, we developed an accelerated Y-shaped DNA catalytic (YDC) circuit for high-efficiency intracellular imaging of microRNA. By grafting the catalytic hairpin assembly (CHA) reactants into an integrated Y-shaped scaffold, the CHA probes were concentrated in a compact space, thus achieving high signal amplification. Profiting from the spatially confined reaction and the self-sustainably assembled DNA products, the YDC system facilitated reliable and in situ microRNA imaging in live cells. Compared with the homogeneously dispersed CHA reactants, the integrated YDC system could efficiently promote the reaction kinetics as well as the uniform delivery of CHA probes, thus providing a robust and reliable analytical tool for disease diagnosis and monitoring.

A Cationic Copolymer Enhances Responsiveness andRobustness ofDNA Circuits.

Toehold-mediated DNA circuits are extensively employed to construct diverse DNA nanodevices and signal amplifiers. However, operations of these circuits are slow and highly susceptive to molecular noise such as the interference from bystander DNA strands. Herein, this work investigates the effects of a series of cationic copolymers on DNA catalytic hairpin assembly, a representative toehold-mediated DNA circuit. One copolymer, poly(L -lysine)-graft-dextran, significantly enhances the reaction rate by 30-fold due to its electrostatic interaction with DNA. Moreover, the copolymer considerably alleviates the circuit's dependency on the length and GC content of toehold, thereby enhancing the robustness of circuit operation against molecular noise. The general effectiveness of poly(L -lysine)-graft-dextran is demonstrated through kinetic characterization of a DNA AND logic circuit. Therefore, use of a cationic copolymer is a versatile and efficient approach to enhance the operation rate and robustness of toehold-mediated DNA circuits, paving the way for more flexible design and broader application.

A Cellular Membrane‐Confined Concatenate DNA Circuit for Non‐Invasive Cell Modulation with High Accuracy and Efficiency

AbstractNon‐invasive cell regulation represents a promising approach for on‐site cell modulation, but is confronted with inaccuracy or poor cell selectivity. Herein, by virtue of the interfacial‐activated concatenate DNA circuit, an activated recombination‐to‐modulation (ARM) machinery is developed to achieve accurate membrane imaging and effective cell modulation. Bioorthogonal signal transduction through the catalytic hairpin assembly circuit can continuously regenerate triggers for activating the subsequent circuit, with no activator consumption and occupation. The ARM strategy utilizes the amplified hybridization chain reaction to sensitively rearrange membrane receptors for blocking the related signaling pathways and indirectly modulating cell behaviors. Based on these two membrane receptor inputs, the controllably‐activated ARM strategy enables the identification of target cells among similar interfering cells with high accuracy and sensitivity. This machinery has demonstrated good performance in the efficient inhibition of tumor metastasis via the disruption of HGF/c‐Met‐signaling pathway. The modular‐designed ARM machinery provides a general platform for non‐invasive and specific cell modulation, suggesting a broad opportunity for advanced exploration of cellular metabolism behaviors.

N/S-Co-doped carbon dot-based FRET ratiometric fluorescence aptasensing platform modulated with entropy-driven DNA amplifier for ochratoxin A detection.

This study proposes a nitrogen and sulfur co-doped carbon dot (N/S-CD)-based FRET ratiometric fluorescence aptasensing strategy modulated with entropy-driven DNA amplifier for sensitive and accurate detection of ochratoxin A (OTA). In the strategy, a duplex DNA probe containing OTA aptamer and complementary DNA (cDNA) is designed as arecognition and transformation element. Upon sensing of target OTA, the cDNA was liberated, and triggered a three-chain DNA composite-based entropy-driven DNA circuit amplification, making CuO probes anchor on amagnetic bead (MB). The CuO-encoded MB complex probe is finally turned into abundant Cu2+, which oxidizes o-phenylenediamine (oPD) to generate 2,3-diaminophenazine (DAP) with yellow fluorescence and further triggers FRET between the blue fluorescent N/S-CDs and DAP. The changes in ratiometric fluorescence are related to the OTA concentration. Originating from the synergistic amplifications from the entropy-driven DNA circuits and Cu2+ amplification, the strategy dramatically enhanced detection performance. A limit of detection as low as 0.006 pg/mL of OTA was achieved. Significantly, the aptasensor can visually evaluate the OTA via on-site visual screening. Moreover, the high-confidence quantification of the OTA in real samples with results consistent with that of the LC-MS method indicated that the proposed strategy has practical application prospects for sensitive and accurate quantification in food safety.

Programming DNA Circuits for Controlled Immunostimulation through CpG Oligodeoxynucleotide Delivery.

Herein, we present a DNA circuit programmed for the delivery of CpG oligodeoxynucleotides (CpG ODNs) with the pharmacological immunostimulation function. The circuit employs a complementary DNA (cDNA) strand to deactivate the biological function of CpG ODNs via hybridization, while T7 exonuclease mediates the activation by hydrolyzing the cDNA and releasing the CpG ODN as an active moiety. We investigated the influence of several factors on the kinetic profile and temporal behavior of the circuit. These include the design of the cDNA strand, the concentration of the DNA duplex, and the concentration of T7 exonuclease. The DNA circuit's in vitro activation resulted in toll-like receptor 9 stimulation in the HEK-engineered cell line, as well as tumor necrosis factor-alpha release by J774A.1 macrophages. By programming the DNA circuit to control the release of the CpG ODN, we achieved an altered pharmacological profile with acute and potent immunostimulation, in comparison to a system without controlled CpG ODN release, which exhibited a slow and delayed response. Our findings demonstrate the potential of DNA circuits in controlling the pharmacological activity of DNA strands for controlled drug delivery.

Accelerating Cell-Free Biosensors by Entropy-Driven Assembly of Transcription Templates.

Due to its high efficiency and selectivity, cell-free biosynthesis has found broad utility in the fields of bioproduction, environment monitoring, and disease diagnostics. However, the practical application is limited by its low productivity. Here, we introduce the entropy-driven assembly of transcription templates as dynamic amplifying modules to accelerate the cell-free transcription process. The catalytic DNA circuit with high sensitivity and enzyme-free format contributes to the production of large amounts of transcription templates, drastically accelerating the as-designed cell-free transcription system without interference from multiple enzymes. The proposed approach was successfully applied to the ultrasensitive detection of SARS-CoV-2, improving the sensitivity by 3 orders of magnitude. Thanks to the high programmability and diverse light-up RNA pairs, the method can be adapted to multiplexing detection, successfully demonstrated by the analysis of two different sites of the SARS-CoV-2 gene in parallel. Further, the flexibility of the entropy-driven circuit enables a dynamic responding range by tuning the circuit layers, which is beneficial for responding to targets with different concentration ranges. The strategy was also applied to the analysis of clinical samples, providing an alternative for sensitively detecting the current SARS-CoV-2 RNA that quickly mutates.

Cascaded, Feedback-Driven, and Spatially Localized Emergence of Constitutional Dynamic Networks Driven by Enzyme-Free Catalytic DNA Circuits.

The enzyme-free catalytic hairpin assembly (CHA) process is introduced as a functional reaction module for guided, high-throughput, emergence, and evolution of constitutional dynamic networks, CDNs, from a set of nucleic acids. The process is applied to assemble networks of variable complexities, functionalities, and spatial confinement, and the systems provide possible mechanistic pathways for the evolution of dynamic networks under prebiotic conditions. Subjecting a set of four or six structurally engineered hairpins to a promoter P1 leads to the CHA-guided emergence of a [2 × 2] CDN or the evolution of a [3 × 3] CDN, respectively. Reacting of a set of branched three-arm DNA-hairpin-functionalized junctions to the promoter strand activates the CHA-induced emergence of a three-dimensional (3D) CDN framework emulating native gene regulatory networks. In addition, activation of a two-layer CHA cascade circuit or a cross-catalytic CHA circuit and cascaded driving feedback-driven evolution of CDNs are demonstrated. Also, subjecting a four-hairpin-modified DNA tetrahedron nanostructure to an auxiliary promoter strand simulates the evolution of a dynamically equilibrated DNA tetrahedron-based CDN that undergoes secondary fueled dynamic reconfiguration. Finally, the effective permeation of DNA tetrahedron structures into cells is utilized to integrate the four-hairpin-functionalized tetrahedron reaction module into cells. The spatially localized miRNA-triggered CHA evolution and reconfiguration of CDNs allowed the logic-gated imaging of intracellular RNAs. Beyond the bioanalytical applications of the systems, the study introduces possible mechanistic pathways for the evolution of functional networks under prebiotic conditions.

Concatenated dynamic DNA network modulated SERS aptasensor based on gold-magnetic nanochains and Au@Ag nanoparticles for enzyme-free amplification analysis of tetracycline

Tetracycline (TC) poses a great threat to food and environmental safety due to its misuse in animal husbandry and aquaculture. Therefore, an efficient analytical method is needed for the detection of TC to prevent possible hazards. Herein, a cascade amplification SERS aptasensor for sensitive determination of TC was constructed based on aptamer, enzyme-free DNA circuits, and SERS technology. The capture probe and signal probe were obtained by binding DNA hairpins H1 and H2 to the prepared Fe3O4@hollow-TiO2/Au nanochains (Fe3O4@h-TiO2/Au NCs) and Au@4-MBA@Ag nanoparticles, respectively. The dual amplification of EDC-CHA circuits significantly facilitated the sensitivity of the aptasensor. Additionally, the introduction of Fe3O4 simplified the operation of the sensing platform due to its superb magnetic capability. Under optimal conditions, the developed aptasensor exhibited a distinct linear response to TC with a low limit of detection of 15.91 pg mL−1. Furthermore, the proposed cascaded amplification sensing strategy exhibited excellent specificity and storage stability, and its practicability and reliability were verified by TC detection of real samples. This study provides a promising idea for the development of specific and sensitive signal amplification analysis platforms in the field of food safety.

Entropy-driven DNA circuit with two-stage strand displacement for elegant and robust detection of miRNA let-7a

MicroRNAs (miRNAs) research in cancer diagnosis is expanding, on account of miRNAs were demonstrated to be key indicator of gene expression and hopeful candidates for biomarkers. In this study, a stable miRNA-let-7a fluorescent biosensor was successfully designed based on an exonuclease Ⅲ-assisted two-stage strand displacement reaction (SDR). First, an entropy-driven SDR containing a three-chain structure of the substrate is used in our designed biosensor, leading to reduce the reversibility of the target recycling process in each step. The target acts on the first stage to start the entropy-driven SDR, which generates the trigger used to stimulate the exonuclease Ⅲ-assisted SDR in the second stage. At the same time, we design a SDR one-step amplification strategy as a comparison. Expectly, this developed two-stage strand displacement system has a low detection limit of 25.0 pM as well as a broad detection range of 4 orders of magnitude, making it more sensitive than the SDR one-step sensor, whose detection limit is 0.8 nM. In addition, this sensor has high specificity across members of the miRNA family. Therefore, we can take advantage of this biosensor to promote miRNA research in cancer diagnosis sensing systems.

Triplex-structure based DNA circuits with ultra-low leakage and high signal-to-noise ratio

DNA circuits are powerful tools in various applications such as logical computation, molecular diagnosis and synthetic biology. Leakage is a major problem in constructing complex DNA circuits. It directly affects the output signal and harms the circuit's performance significantly. In the traditional DNA circuits, the gate complex is a duplex structure. There are insufficient energy barriers to prevent spontaneous detachment of strands, resulting in a leak prone. Herein, we have developed triplex-structure based DNA circuit with ultra-low leakage and high signal-to-noise ratio (SNR). The triplex structure improves the stability in the absence of input. At the same time, the driving force of the strand displacement cascades reduces the influence of the triplex structure on the desired reaction. The SNR of the DNA circuit was increased to 695, while the desired reaction rate remained 90% of the conventional translator circuit. The triplex-structure mediated leakage prevention strategy was further tested at different temperatures and in DNA translator and seesaw circuits. We also constructed modular basic logic gates with a high efficiency and low leakage. On this basis, we further constructed triplex-structure based tertiary DNA logic circuits, and the SNR reached 295, which, to the best of our knowledge, was among the highest of the field. We believe that our scheme provides a novel, valid, and general tool for reducing leakages, and we anticipate that it will be widely adopted in DNA nanotechnology.

“Repaired and initiated” intramolecular DNA circuit enables the amplified imaging of DNA repair enzyme activity in live cells

Intramolecular catalytic hairpin assembly (intraCHA) has recently been developed. However, non-nucleic acid-initiated intraCHA is rarely explored. The only protein-initiated intraCHA depends on the unwarrantable binding affinity between protein and its aptamer, which generally results in limited initiation efficiency. Herein, a “repaired and initiated” intraCHA (RI-intraCHA) nanosystem was designed and its operation was reported for amplified imaging of DNA repair enzyme activity in live cells. 8-oxoguanine (8-OG) DNA glycosylase was selected as the model DNA repair enzyme. The RI-intraCHA nanosystem was constructed by linking a dsDNA (containing 8-OG sites and trigger strand), two hairpins to different vertexes of DNA tetrahedron. The nanosystem could independently enter into live cells in virtue of cell permeability of DNA tetrahedron. Upon repair action of endogenous 8-OG DNA glycosylase, intraCHA reaction in the nanosystem was initiated efficiently, generating amplified fluorescence signal. The nanosystem provided a detection limit of 0.2443 U/mL for 8-OG DNA glycosylase, which was lower than that of the reported imaging approaches. Furthermore, the nanosystem exhibited satisfactory biosafety and biostability, achieving amplified imaging of intracellular 8-OG DNA glycosylase activity. The designed RI-intraCHA nanosystem provided a promising tool for the research of basic biology of intracellular DNA repair enzymes as well as their clinic correlations.