Nucleic Acid Circuits Research Articles

Lego-Like Catalytic Hairpin Assembly Enables Controllable DNA-Oligomer Formation and Spatiotemporal Amplification in Single Molecular Signaling.

While the solid-state nanopore shows increasing potential during sensitive and label-free single molecular analysis, target concentration and signal amplification method is in urgent need. In this article, a solution via designing a model nucleic acid circuit reaction that can produce "Y" shape-structure three-way DNA oligomers with controllable size and polymerization degree is proposed. Such a so-called lego-like three-way catalytic hairpin assembly (LK-3W-CHA) can provide both concentration amplification (via CHA circuit) and programmable size control (via lego-like building mode) to enhance spatiotemporal resolution in single molecular sensing of solid-state nanopore. Oligomers containing 1-4 DNA three-way junctions (Y monomers, Y1-Y4) are designed in proof-of-concept experiments and applications. When the oligomers are applied to direct translocation measurements, Y2-Y4 can significantly increase the signal resolution and stability than that of Y1. Meanwhile, Y1 to Y4 can be used as the tags on the long DNA carrier to provide very legible secondary signals for specific identification, multiple assays, and information storage. Compared with other possible tags, Y1-Y4 provides higher signal density and amplitude, and quasi-linear "inner reference" for each other, which may provide more systematic, reliable, and controllable experimental results.

Automated Leak Analysis of Nucleic Acid Circuits.

Nucleic acids are a powerful engineering material that can be used to implement a broad range of computational circuits at the nanoscale, with potential applications in high-precision biosensing, diagnostics, and therapeutics. However, nucleic acid circuits are prone to leaks, which result from unintended displacement interactions between nucleic acid strands. Such leaks can grow combinatorially with circuit size, are challenging to mitigate, and can significantly compromise circuit behavior. While several techniques have been proposed to partially mitigate leaks, computational methods for designing new leak mitigation strategies and comparing their effectiveness on circuit behavior are limited. Here we present a general method for the automated leak analysis of nucleic acid circuits, referred to as DSD Leaks. Our method extends the logic programming functionality of the Visual DSD language, developed for the design and analysis of nucleic acid circuits, with predicates for leak generation, a leak reaction enumeration algorithm, and predicates to exclude low probability leak reactions. We use our method to identify the critical leak reactions affecting the performance of control circuits, and to analyze leak mitigation strategies by automatically generating leak reactions. Finally, we design new control circuits with substantially reduced leakage including a sophisticated proportional-integral controller circuit, which can in turn serve as building blocks for future circuits. By integrating our method within an open-source nucleic acid circuit design tool, we enable the leak analysis of a broad range of circuits, as an important step toward facilitating robust and scalable nucleic acid circuit design.

A functional RNA/DNA circuit for one-pot detection of SARS-CoV-2 RNA.

A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a functional RNA/DNA circuit. By ingeniously integrating the nucleic acid circuit technology and CRISPR/cas12a system, this method can achieve femtomolar detection of the target RNA in one step and successfully distinguish COVID-19 positive cases from clinical samples, proving its great potential for clinical application.

Highly Sensitive Detection of miR-21 through Target-Activated Catalytic Hairpin Assembly of X-Shaped DNA Nanostructures.

MicroRNAs (miRNAs) are found in extremely low concentrations in cells, so highly sensitive quantitation is a great challenge. Herein, a simple dual-amplification strategy involving target-activated catalytic hairpin assembly (CHA) coupled with multiple fluorophores concentrated on one X-shaped DNA is reported. In this strategy, four hairpin probes (H1, H2, H3, and H4) are modified with FAM and BHQ1 at both sticky ends, while a circulating hairpin probe (H0) is used to activate CHA circuits once it binds to complementary sequences in the target miR-21 (T). The powerful dual-amplification cascades in Förster resonance energy transfer (FRET)-based nonenzymatic nucleic acid circuits are triggered by T-H0-activated formation of the X-shaped DNA nanostructure, freeing T-H0 for the next CHA reaction cycle. CHA circuits increase the fluorescence due to the wide distance between FAM and BHQ1 in the formed X-shaped DNA nanostructure, resulting in signal amplification and highly sensitive detection of miR-21, with a limit of detection (LOD, 3σ) of 0.025 nM, which is 25.6 or 57.6 times lower than that obtained through a single-amplification strategy without multiple fluorophores on one X-shaped DNA or CHA circuit. Furthermore, this cascade reaction was completed in 45 min, effectively avoiding target degradation. This new enzyme-free signal amplification strategy holds promising potential for sensitively detecting different DNA or RNA sequences by simply adapting the fragment of the H0 sequence complementary to the target.

Proximity binding induced nucleic acid cascade amplification strategy for ultrasensitive homogeneous detection of PSA

In this work, based on the powerful cycle amplification cascades of proximity hybridization-induced hybridization chain reaction and catalyzed hairpin assembly, we engineered a nonenzymatic and ultrasensitive method which combined the Mg2+-DNAzyme recycling signal amplification for the analysis of the human prostate specific antigen. Herein, we adopted PSA-conjugates as triggers in the self-assembly process of two hairpin DNAs (H1, H2) into the products of the CHA which could activate the HCR to induce repeated hybridization. And both ends of each adjacent sequence of the HCR products could form a unit of Mg2+-DNAzyme which in presence of cofactor Mg2+ could recognize and cyclically cleave the hairpin probes in the solution and thus generate observably enhanced fluorescent signal. Benefit from the nucleic acid circuit amplification strategy, PSA of concentration low to 0.73 pg mL−1 was detected in this system. This homogeneous sensing method in solution avoid the use of the sophisticated equipment and complex operation, as well as addition of artificial enzyme, thus greatly reducing the constraints and complexity of experimental conditions. Moreover, considering most protein biomarkers in serum don't have their corresponding aptamers, this sensing method provide a general sensing approach for homogeneous sensitive detection of these important protein biomarkers which transfer rough antigen-antibody interactivity to smart signal amplification sensing strategies, thus exhibiting a remarkable prospect in clinical application.

A kinetically controlled platform for ligand-oligonucleotide transduction

Ligand-oligonucleotide transduction provides the critical pathway to integrate non-nucleic acid molecules into nucleic acid circuits and nanomachines for a variety of strand-displacement related applications. Herein, a general platform is constructed to convert the signals of ligands into desired oligonucleotides through a precise kinetic control. In this design, the ligand-aptamer binding sequence with an engineered duplex stem is introduced between the toehold and displacement domains of the invading strand to regulate the strand-displacement reaction. Employing this platform, we achieve efficient transduction of both small molecules and proteins orthogonally, and more importantly, establish logical and cascading operations between different ligands for versatile transduction. Besides, this platform is capable of being directly coupled with the signal amplification systems to further enhance the transduction performance. This kinetically controlled platform presents unique features with designing simplicity and flexibility, expandable complexity and system compatibility, which may pave a broad road towards nucleic acid-based developments of sophisticated transduction networks.

A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics.

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas-powered catalytic nucleic acid circuit, namely, CRISPR-Cas-only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer-associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

Processing DNA-Based Molecular Signals into Graphical Displays.

DNA is now well-established as a nanoscale building material with applications in fields such as biosensing and molecular computation. Molecular processes such as logic gates, nucleic acid circuits, and multiplexed detection have used different readout strategies to measure the output signal. In biosensing, this output can be the diagnosis of a disease biomarker, whereas in molecular computation, the output can be the result of a mathematical operation carried out using DNA. Recent developments have shown that the output of such processes can be displayed graphically as a macroscopic symbol or an alphanumeric character on multiwell plates, microarray chips, gels, lateral flow devices, and DNA origami surfaces. This review discusses the concepts behind such graphical readouts of molecular events, available display platforms, and the advantages and challenges in adapting such methods for practical use. Graphical display systems have the potential to be used in the creation of intelligent computing and sensing devices by which nanoscale binding events are translated into macroscopic visual readouts.

One-tube smart genetic testing via coupling isothermal amplification and three-way nucleic acid circuit to glucometers

Urgent demand for portable diagnosis has promoted a new sensing strategy that uses personal glucometer (PGM) to detect non-glucose targets. Even though great progresses have been achieved in terms of target range and sensing principle, issues such as low final signal-to-background ratio and hard-to-realize one-tube smart analysis still exist and challenge real-world applications in gene detection. Here we propose a practical solution via coupling isothermal amplification (i.e. LAMP) and three-way amplifiable catalytic hairpin assembly (i.e. CHA) to a PGM. It allows direct transduction from genomic information to commercial portable devices with all of ultra-high sensitivity, specificity and enhanced signal-to-noise ratio. Compared with previous report without signal amplification, the introduction of CHA has successfully improved the signal amplitude by at least 12.5 folds. More importantly, through importing an effective three-way junction based transduction, we also innovatively develop a one-tube logical or multiplex analysis strategy in PGM based detection. Totally four situations of two foodborne bacteria genes, in Cronobacter sakazakii (ompA) and Escherichia coli (malB), could be directly readout using the final PGM signals, with the lowest detection amount down to less than 100 molecular copies (6.6 × 10−18 M). It is believed such a LAMP-CHA-PGM method has been already sensitive, specific, and of great potential for practically portable gene diagnostics.

Double-signal mode based on metal-organic framework coupled cascaded nucleic acid circuits for accurate and sensitive detection of serum circulating miRNAs.

The tough challenges for the sensitive and accurate detection of circulating miRNAs (cmiRNAs) in the peripheral blood are low abundance, and high interference from environmental or background factors or other molecules. Here, we developed a double-signal mode based on metal-organic framework coupled cascaded nucleic acid circuits for the accurate and sensitive detection of serum circulating miRNAs.

Heterochiral DNA Strand-Displacement Based on Chimeric d/l-Oligonucleotides.

Heterochiral DNA strand-displacement reactions enable sequence-specific interfacing of oligonucleotide enantiomers, making it possible to interface native d-nucleic acids with molecular circuits built using nuclease-resistant l-DNA. To date, all heterochiral reactions have relied on peptide nucleic acid (PNA), which places potential limits on the scope and utility of this approach. Herein, we now report heterochiral strand-displacement in the absence of PNA, instead utilizing chimeric d/l-DNA complexes to interface oligonucleotides of the opposite chirality. We show that these strand-displacement reactions can be easily integrated into multicomponent heterochiral circuits, are compatible with both DNA and RNA inputs, and can be engineered to function in serum-supplemented medium. We anticipate that these new reactions will lead to a wider application of heterochiral strand-displacement, especially in the design of biocompatible nucleic acid circuits that can reliably operate within living systems.

A signal-flexible gene diagnostic strategy coupling loop-mediated isothermal amplification with hybridization chain reaction

Recent study proves that the combination of loop mediated isothermal nucleic acid amplification (LAMP) with one-step strand displacement (OSD) is of great help to improve the sequence specificity during genetic detection. However, because OSD is incapable of signal amplification, the signal-to-noise ratio or the observable signal change may be usually not significant enough to satisfy practical usage. With the purpose to overcome this challenge, herein a more advanced and practical sensing principle is developed with the OSD replaced by an amplifiable nucleic acid circuit, hybridization chain reaction (HCR). The very contagious norovirus (NoV) was employed as the model target. Compared with LAMP-OSD, the LAMP-HCR can detect as few as 30 copies of NoV gene in 2% fecal samples with significantly enlarged signal change and signal-to-background ratio. Therefore, more reliable detection is achieved. Moreover, due to the high compatibility of HCR, the final LAMP-HCR products can be flexibly transduced into different types of readouts, including fluorescence, flow cytometer (FCM) and even a personal glucose meter (PGM). This further enlarges the operating environments for the detection from hospital labs, bedsides, to potential off-the-shelf devices in local pharmacies. Especially when using FCM or PGM, with the assistance of magnetic beads (MBs), the detection shows even higher tolerance capability to complicated biological matrices.

Autonomous dynamic control of DNA nanostructure self-assembly.

Biological cells routinely reconfigure their shape using dynamic signalling and regulatory networks that direct self-assembly processes in time and space, through molecular components that sense, process and transmit information from the environment. A similar strategy could be used to enable life-like behaviours in synthetic materials. Nucleic acid nanotechnology offers a promising route towards this goal through a variety of sensors, logic and dynamic components and self-assembling structures. Here, by harnessing both dynamic and structural DNA nanotechnology, we demonstrate dynamic control of the self-assembly of DNA nanotubes-a well-known class of programmable DNA nanostructures. Nanotube assembly and disassembly is controlled with minimal synthetic gene systems, including an autonomous molecular oscillator. We use a coarse-grained computational model to capture nanotube length distribution dynamics in response to inputs from nucleic acid circuits. We hope that these results may find use for the development of responsive nucleic acid materials, with potential applications in biomaterials science, nanofabrication and drug delivery.

Pattern Generation with Nucleic Acid Chemical Reaction Networks.

Biological and biochemical systems are manifestations of chemical reaction networks (CRNs). The ability to design and engineer such networks may allow the construction of artificial systems that are as complex as those seen in biology, opening the way to translational possibilities including adaptive materials. One venue for progress is the design of autonomous systems capable of pattern generation; however many synthetic CRNs, such as the Belousov-Zhabotinsky reaction, cannot be rewired to encode more complex interactions and thus lack the capacity for more detailed engineering algorithms. In contrast, DNA is an information-rich molecule with predictable and reliable base-pairing interactions and well-studied kinetics, and the use of DNA has greatly enabled the rational design of much more complex synthetic CRNs. Recent advances in the DNA computing field include circuits for pattern transformation, an example of self-organization. An arsenal of tools for designing DNA circuits to implement various CRNs has been developed by DNA nanotechnologists, including software to reliably program strand-displacement nucleic acid circuits. In addition, DNA walkers can be used to create CRNs with controlled diffusivity, while DNA gels similarly represent a new medium for implementing CRNs that may ultimately lead to the development of smart materials. As we will argue, future endeavors in nucleic acid-based pattern generation will be most greatly advanced by harnessing well-known enzymatic processes to serve as generators and amplifiers. Once nucleic acid computing tools are further developed to expedite the design process of pattern generation, we anticipate a transition from proof-of-concept curiosities to application-driven inquiries.

Cancer-Specific MicroRNA Analysis with a Nonenzymatic Nucleic Acid Circuit.

Sensitive detection of gastric cancer-related biomarkers in human serum provides a promising means for early cancer diagnosis. Herein, we report the design of a nucleic acid circuit for gastric cancer-related microRNA-27a (miRNA-27a) detection based on dual toehold-mediated circular strand displacement amplification (CSDA). In the presence of miRNA-27a, the hybridization between miRNA-27a and probe DNA on magnetic beads through toehold 1 leads to the release of fluorescent DNA and the exposure of a new toehold 2 on linker DNA. After hybridization with catalytic DNA, CSDA is initiated and target miRNA-27a is released to participate in the next cyclic reaction; therefore, a greatly enhanced fluorescence signal is produced. The efficient magnetic separation makes the sensitive detection of miRNA-27a be accomplished within 45 min. With the efficient CSDA, the detection limit of the system (0.8 pM) is ∼100 folds lower than that of the system based on strand displacement without CSDA (79.3 pM). Furthermore, the system also showed good stability and sensitivity to discriminate single-base mismatch, which allows the detection of miRNA-27a in human serum samples. This study provides a novel platform and approach for the rapid quantitative determination of miRNA, which has great potential in clinical diagnosis and disease treatment.

Thiol-free oligonucleotide surface modification of gold nanoparticles for nanostructure assembly.

Gold nanoparticles (AuNPs) decorated with thiol-modified DNA (HS-DNA) strands are an extensively studied, easily adjustable, and highly controllable material for constructing 3D nanostructures with various shapes and functions. However, few reproducible and robust methods involving DNA templates as a key reagent are available for obtaining 3D nanoparticle assemblies. It is still challenging to strictly control the number and location of DNA strands on the AuNP surface. Here, we introduce an efficient approach for the surface modification of AuNPs using unmodified DNA oligonucleotides by building DNA cages that trap the nanoparticles. This enables us to vary the process of nanostructure assembly and create anisotropic nanoparticles that are necessary for directed structure construction. This developed method simplifies the production process in comparison with conventional HS-DNA modification protocols and helps to precisely control the density and position of functional DNA strands designed for further hybridization with other AuNP conjugates.

Digital quantitative detection of serum circulating miRNAs using dual-enhanced magnetobiosensors based on cascaded nucleic acid circuits.

The tough challenges for the convenient and quantitative determination of circulating miRNAs (cmiRNAs) in the peripheral blood are low abundance, high interference and lack of direct digital readout. Here, we developed dual-enhanced magnetobiosensors based on cascaded nucleic acid circuits, which integrate catalyzed hairpin assembly (CHA) with the hybridization chain reaction (HCR), for sensitive, portable and digital quantitative detection of circulating miRNAs in serum by a personal glucose meter (PGM).

Lighting Up Fluorescent Silver Clusters via Target-Catalyzed Hairpin Assembly for Amplified Biosensing.

Isothermal enzyme-free nucleic acid circuits have been developed for carrying out diverse functions ranging from dictate biocomputing to amplified biosensing. Catalytic hairpin assembly (CHA), the catalyzed cross-opening of two hairpin substrates by an initiator, has attracted increasing attention because of its facile design and high amplification capacity. The complex labeling and frequent photobleaching of a conventional fluorescent CHA biosensor still remains a challenge that needs to be solved. Herein, we constructed a new label-free and enzyme-free isothermal CHA lighting up AgNCs strategy for amplified nucleic acid assay by integrating the interfacially and spatially sensitive feature of DNA-templated fluorescent silver nanoclusters (DNA-AgNCs) and the high signal amplification capability of the CHA circuit. In this strategy, one polyguanine-grafted hairpin and the other AgNCs-capturing hairpin were engineered as assembly constitutes, which were kinetically impeded from cross-hybridizations without target. However, in the presence of target, the CHA-catalyzed assembly of two functional hairpins was successively progressed and concomitantly accompanied by an efficient accommodation of AgNCs to the polyguanine-elongated dsDNA product, leading to highly efficient AgNCs-lighting up and to the generation of an amplified fluorescence signal. As a simple mix-and-detect strategy, the isothermal enzyme-free CHA-mediated lighting up AgNCs (CHA-AgNCs) system provided a facile visualization way for amplified detection of DNA with a detection limit of 20 pM, which was comparable to or even better than some enzyme-involved amplification methods. The homogeneous CHA-AgNCs system can be used as a general sensing platform and be easily adapted for analyzing other biologically important analytes, for example, microRNA (miRNA), by introducing the sensing module consisting of an auxiliary hairpin through an easy-to-integrate procedure. By taking advantage of the signal amplification features of CHA and the robust AgNCs-lighting up procedure, we anticipate that the CHA-lighting up AgNCs system can provide an important tool for biomedicine and bioimaging applications and thus should hold great promise in clinical diagnoses and treatment fields.

Genetically Encoded Catalytic Hairpin Assembly for Sensitive RNA Imaging in Live Cells.

DNA and RNA nanotechnology has been used for the development of dynamic molecular devices. In particular, programmable enzyme-free nucleic acid circuits, such as catalytic hairpin assembly, have been demonstrated as useful tools for bioanalysis and to scale up system complexity to an extent beyond current cellular genetic circuits. However, the intracellular functions of most synthetic nucleic acid circuits have been hindered by challenges in the biological delivery and degradation. On the other hand, genetically encoded and transcribed RNA circuits emerge as alternative powerful tools for long-term embedded cellular analysis and regulation. Herein, we reported a genetically encoded RNA-based catalytic hairpin assembly circuit for sensitive RNA imaging inside living cells. The split version of Broccoli, a fluorogenic RNA aptamer, was used as the reporter. One target RNA can catalytically trigger the fluorescence from tens-to-hundreds of Broccoli. As a result, target RNAs can be sensitively detected. We have further engineered our circuit to allow easy programming to image various target RNA sequences. This design principle opens the arena for developing a large variety of genetically encoded RNA circuits for cellular applications.

Enzyme-free, signal-amplified nucleic acid circuits for biosensing and bioimaging analysis.

Enzyme-free, signal-amplified nucleic acid circuits utilize programmed assembly reactions between nucleic acid substrates to transduce a chemical input into an amplified detection signal. These circuits have shown great potential for developing biosensors for high-sensitivity and high-selectivity detection of varying targets including nucleic acids, small molecules and proteins in vitro and for high-contrast in situ visualization and imaging of these targets in tissues and living cells. We review the background of the enzyme-free, signal-amplified nucleic acid circuits, including their mechanism, significance, types and development. We also review current applications of these circuits for biosensors and bioimaging.