Toehold-mediated Strand Displacement Reaction Research Articles

Size-Discriminative DNA Nanocage Framework Enables Sensitive and High-Fidelity Imaging of Mature MicroRNA in Living Cells.

Mature microRNAs (miRNAs) are closely associated with cell proliferation and differentiation, stress response, and carcinogenesis, and monitoring intracellular miRNAs can contribute to the studies of their regulatory roles and molecular mechanisms of disease progression. However, accurate and reliable detection of mature miRNAs in complex physiological environments encounters the challenge of undesired detection accuracy ascribed to the coexistence of their precursor microRNAs (pre-miRNAs) and degradation of sensing probes. Here, we demonstrate the synthesis of a new size-discriminative DNA nanocage framework (DNF) for the sensitive monitoring of mature miRNA-21 in living cells with high accuracy via cascaded toehold-mediated strand displacement reaction (TSDR) amplifications. The DNF is prepared by a simple self-assembly of six ssDNAs, and the signal probes are docked inside the DNF. Because of its rigid framework structure, the DNF shows enhanced enzyme stability. Upon entering cells, only the short target mature miRNA-21 sequences instead of the large-sized pre-miRNAs are allowed to be accommodated inside the cavity of the DNF owing to the size-discriminative capability of the DNF. The cascaded TSDR amplifications can thus be activated by the mature miRNA-21 together with endogenous ATP to result in magnified fluorescence for sensitive detection and selective discrimination of miRNA-21 from the interference pre-miRNAs. Our results indicate that the DNF probes can offer robust sensing means for detecting various intracellular mature miRNAs with high accuracy for disease diagnoses and biomedical studies.

DNA methylation detection and site analysis by using an electrochemical biosensor constructed based on toehold-mediated strand displacement reaction

DNA methylation has become a novel target for early diagnosis and prognosis of cancer as well as other related diseases. The accurate detection of the methylation sites of specific genes proved to be of great significance. However, the complex biological nature of clinical samples and the detection of low-abundance targets led to higher requirements for the testing technology. It has been found that by virtue of high sensitivity, rapid response, low cost, facile operation and applicability to microanalysis, electrochemical sensors have greatly contributed to the process of clinical diagnosis. In this study, a facile, rapid and highly sensitive electrochemical biosensor based on the peak current change was developed on the basis of high selectivity of toehold and greater efficiency of PNA strand displacement and used for the detection and site analysis of DNA methylation. Moreover, compared with non-methylated DNA sequences, methylated DNA sequences could be readily invaded by PNA probes, thereby resulting in the strand displacement and significant electrical signals. Therefore, methylation of cytosine sites was primarily analyzed based on electrical signals. Strand displacement by the target DNA sequences with different methylated sites can lead to substantial changes of strand displacement efficiency. As a result, the methylation sites can be analyzed on the basis of corresponding peak current response relation. This method has a detection limit of 0.075 pM and does not involve various complicated steps such as bisulfite treatment, enzyme digestion and PCR amplification. Indeed, one detection cycle can be completed in 60 min. The proposed technology might exhibit great potential in early clinical diagnosis and risk assessment of cancers and related diseases.

Upconversion Nanoparticle@Au Core-Satellite Assemblies for In Situ Amplified Imaging of MicroRNA in Living Cells and Combined Cancer Phototherapy.

Stimuli-responsive therapy of cancer with spatial and temporal control is crucial in improving the treatment efficacy and minimizing the side effects. MicroRNA (miRNA) as an important biomarker has become one of the most promising endogenous stimuli for cancer therapy. However, the therapy efficacy is often impeded by the low expression amount of miRNA. Herein, the upconversion nanoparticle@Au (UCNP@Au) core-satellite nanostructures are rationally fabricated for isothermal amplification detection and in situ imaging of microRNA-21 (miR-21) in living cells based on the toehold-mediated strand displacement (TMSD) reaction, which is further applied to miRNA-responsive combined photothermal and photodynamic therapy of breast cancer. The UCNP@Au are constructed by linking AuNPs to photosensitizers Rose Bengal (RB)-loaded UCNPs through DNA hybridization. The upconversion luminescence (UCL) is quenched by AuNPs, resulting in the attenuation of singlet oxygen generation of RB. Once UCNP@Au are internalized into MCF-7 cells, the overexpressed intracellular miR-21 trigger the cyclic disassembly of UCNP@Au through cascade TMSD reactions, which facilitate the restoration of UCL for in situ imaging of miR-21 with signal amplification. Moreover, the released AuNPs are aggregated for photothermal therapy (PTT), while the singlet oxygen generated by RB is enhanced for photodynamic therapy (PDT). Compared with single-mode therapy, the miRNA-activated combinational phototherapy has demonstrated a greatly improved therapeutic efficacy for breast cancer. Therefore, our proposed core-satellite nanostructures cannot only achieve in situ amplified imaging of endogenous miRNA but also provide an effective nanoplatform for stimuli-responsive combinational phototherapy, which hold great prospects in early diagnosis and treatment of cancers.

Digital-resolution and highly sensitive detection of multiple exosomal small RNAs by DNA toehold probe-based photonic resonator absorption microscopy.

Small noncoding RNAs (snRNA) have been emerging as promising diagnostic biomarkers for detecting early stage cancer. Currently existing methods for snRNA detection, including northern blot, reverse transcription-polymerase chain reaction, microarrays and RNA-Seq, are limited to time-consuming, low sensitivity, expensive instrumentation or complex analysis of data. Herein, we present a rapid quantitative analysis of multiple liver cancer-associated exosomal snRNA by a nucleic acid toehold probe-based photonic resonator absorption microscopy (PRAM) assay, with digital resolution and high sensitivity. The assay relies on the use of three toehold probe-encoded gold nanoparticles (AuNPs) and addressable photonic crystal (PC) sensing chips. The presence of target snRNA will initiate toehold-mediated strand displacement reactions that trigger the capture of gold particles onto the PC surface, which is subsequently imaged by PRAM for digital counting of detected snRNA molecules. We achieved highly sensitive and selective detection of three snRNA targets in buffer with a 30min assay protocol, with detection limits of 4.56fM, 4.68fM and 0.69 pM. Having confirmed our assay's performance for detection of snRNA targets spiked into exosomal RNA extracts, we demonstrated its capability for quantitative detection of the same targets from patient blood plasma samples. The approach offers a rapid, simple workflow that operates at room temperature with a single step without enzymatic amplification, while the detection instrument can be implemented as a low-cost portable system for point of care environments.

DNA-Based Daisy Chain Rotaxane Nanocomposite Hydrogels as Dual-Programmable Dynamic Scaffolds for Stem Cell Adhesion.

Interlocked DNA nanostructures perform programmable movements in nanoscales such as sliding, contraction, and expansion. However, utilizing nanoscaled interlocked movements to regulate the functions of larger length scaled matrix and developing their applications has not yet been reported. Herein we describe the assembly of DNA-based daisy chain rotaxane nanostructure (DNA-DCR) composed of two hollow DNA nanostructures as macrocycles, two interlocked axles and two triangular prism-shaped DNA structures as stoppers, in which three mechanical states─fixed extended state (FES), sliding state (SS), and fixed contracted state (FCS)─are characterized by using toehold-mediated strand displacement reaction (SDR). The DNA-DCRs are further used as nanocomposites and introduced into hydrogel matrix to produce interlocked hydrogels, which shows modulable stiffness by elongating the interlocked axles to regulate the hydrogel swelling with hybridization chain reaction (HCR) treatment. Then the DCR-hydrogels are employed as dynamic biointerfaces for human mesenchymal stem cells (hMSCs) adhesion studies. First, hMSCs showed lower cell density on bare DCR-hydrogel treated with HCR-initiated swelling for stiffness decreasing. Second, the cell adhesion ligand (RGD) modified DNA-DCRs are constructed for hydrogel functionalization. DCR(RGD) hydrogel endows the mobility of RGDs by switching the mechanical states of DNA-DCR. HMSCs showed increased cell density on DCRSS(RGD) hydrogel than on DCRFCS(RGD) hydrogel. Therefore, our DNA-DCR nanocomposite hydrogel exhibit dual-programmable performances including swelling adjustment and offering sliding for incorporated ligands, which can be both utilized as dynamic scaffolds for regulating the stem cell adhesion. The dual-programmable cross-scale regulation from interlocked DNA nanostructures to hydrogel matrix was achieved, demonstrating a new pathway of DNA-based materials.

Dynamic modulation of enzyme activity by synthetic CRISPR-Cas6 endonucleases.

In nature, dynamic interactions between enzymes play a crucial role in defining cellular metabolism. By controlling the spatial and temporal organization of these supramolecular complexes called metabolons, natural metabolism can be tuned in a highly dynamic manner. Here, we repurpose the CRISPR-Cas6 family proteins as a synthetic strategy to create dynamic metabolons by combining the ease of RNA processing and the predictability of RNA hybridization for protein assembly. By disturbing RNA-RNA networks using toehold-mediated strand displacement reactions, on-demand assembly and disassembly are achieved using both synthetic RNA triggers and mCherry messenger RNA. Both direct and 'Turn-On' assembly of the pathway enzymes tryptophan-2-monooxygenase and indoleacetamide hydrolase can enhance indole-3-acetic acid production by up to ninefold. Even multimeric enzymes can be assembled to improve malate production by threefold. By interfacing with endogenous mRNAs, more complex metabolons may be constructed, resulting in a self-responsive metabolic machinery capable of adapting to changing cellular demand.

Electrogenerated chemiluminescence biosensor for microRNA detection incorporating enzyme-free dual DNA cyclic amplification and Ru(bpy)32+-functionalized metal-organic framework

Herein, a novel electrogenerated chemiluminescence (ECL) biosensor for the determination of microRNA was developed on the basis of enzyme-free dual signal amplification and Ru(bpy)32+-functionalized metal-organic frameworks (Ru@MIL-101(Al)–NH2) as ECL signal labels. MicroRNA-21 (miRNA-21) was used as a model target, which initiated the toehold-mediated strand displacement reaction (TSDR). The first cyclic amplification TSDR converted a small quantify of miRNA-21 to abundant report strands (RS), which then catalyzed the second cyclic amplification catalytic hairpin assembly (CHA). The CHA ran cyclically to produce numerous H1–H2 duplexes with the exposed segment. Finally, the newly exposed segment of H2 could bind with the ECL probe (DNA-Ru@MIL-101(Al)–NH2), leading to an evident ECL signal enhancement. The results showed that the ECL intensity varied linearly with the logarithm of the concentration of miRNA-21 ranging from 0.01 to 10,000 pM. The detection limit was as low as 4 fM because of the signal amplification through TSDR and CHA processes coupling with Ru@MIL-101(Al)–NH2. In addition, this biosensor has been successfully applied to detect miRNA-21 in MCF-7 cells, providing great potential for the related clinical diagnosis.

Single-Molecule Resettable DNA Computing via Magnetic Tweezers.

DNA-based Boolean logic computing has emerged as a leading technique in biosensing, diagnosis, and therapeutics. Due to the development of the biological and chemical methods, especially the toehold-mediated DNA strand displacement (TMSD) reaction, different logic gates as well as circuits can be performed. However, most of these methods have been conducted at the bulk level, which may lead to missing information and be less controllable. Herein, we engineered single-molecule DNA computing controlled by stretching forces using magnetic tweezers. By tracking the real-time signals of the DNA extension, the output can be determined at a single base-pair resolution. A kinetics-controllable TMSD reaction was realized in the range of a ∼19-fold change of the reaction rate by different stretching forces. OR, AND, and NOT gates were also achieved. In addition, resettable DNA computing using force stretching cycles has been further exemplified. Overall, such a real-time, label-free, and force-controlled single-molecule DNA computing system provided new insight into molecular computing.

Molecular dynamics simulation-guided toehold mediated strand displacement probe for single-nucleotide variants detection.

Single nucleotide variant (SNV) has become an emerging biomarker for various diseases such as cancers and infectious diseases. Toehold-mediated strand displacement (TMSD), the core reaction of DNA nanotechnology, has been widely leveraged to identify SNVs. However, inappropriate choice of mismatch location results in poor discrimination ability. Here, we comprehensively investigate the effect of mismatch location on TMSD kinetics by molecular dynamic simulation tool oxDNA through umbrella sampling and forward flux sampling disclosing that mismatches at the border of the toehold and branch migration domain yield the lowest TMSD reaction rate. Nine disease-related SNVs (SARS-CoV-2-D614G, EGFR-L858R, EGFR-T790M, KRAS-G12R, etc.) were tested experimentally showing a good agreement with simulation. The best choice of mismatch location enables high discrimination factor with a median of 124 for SNV and wild type. Coupling with a probe-sink system, a low variant allele frequency of 0.1% was detected with 3 S/N. We successfully used the probes to detect SNVs with high confidence in the PCR clones of constructed plasmids. This work provides mechanistic insights into TMSD process at the single-nucleotide level and can be a guidance for the design of TMSD system with fine-tuning kinetics for various applications in biosensors and nanotechnology.

Color-Changing Fluorescent Barcode Based on Strand Displacement Reaction Enables Simple Multiplexed Labeling.

Fluorescence imaging techniques have contributed to our understanding of various biological phenomenon; however, fluorescence spectral overlap significantly restricts multiplexing capability. Several strategies have been reported to overcome this limitation by utilizing the superior programmability of DNA technologies and nanostructures, but in practice, it remains challenging to achieve broad adoption of these multiplexed detection methods due to the complexities of these DNA designs. Here we report a color-changing fluorescent barcode (CCFB) approach that enables multiple labeling with simple and small nucleic acid structure design based on sequential toehold-mediated strand displacement reaction. The emission color of CCFB can vary in the predetermined sequence so that multiple targets can be detected simultaneously. The CCFB complex is composed of several oligonucleotides, and its color sequence can be easily expanded further. The CCFB approach is easy and time-saving to operate since the irreversible color-changing reaction occurs by simply adding complementary oligonucleotide. We herein developed 27 different CCFB labels, which required only 14 oligonucleotides. We demonstrated that the CCFB system can be used to label multiple targets by attaching CCFB label to polystyrene beads. Moreover, the CCFB can be used to detect intracellular proteins simultaneously when it is attached to antibodies. We expect that this practical platform will be adopted for comprehensive biomolecular imaging in cells.

Highly sensitive and label-free detection of DILI microRNA biomarker via target recycling and primer exchange reaction amplifications

MicroRNAs (miRNA) are closely associated with the development of diseases in life processes, and the sensitive detection of microRNA biomarkers is beneficial to disease diagnosis and treatment at early stage. Herein, by coupling the target sequence recycling with the primer exchange reaction (PER), a highly sensitive and non-label approach is constructed to detect miRNA-122, the biomarker for drug-induced liver injury. The target sequence cyclically displaces two hairpins on the DNA track via toehold-mediated strand displacement reactions with the assistance of the fuel DNA. The released hairpins further bind the primer to trigger the polymerase-aided PER process for the yield of plenty of G-quadruplex sequences, which then combine with the thioflavin T to drastically enhance its fluorescence for sensing miRNA-122 with a low 49.4 fM detection limit. Highly specific discrimination of the target miRNA-122 can also be realized with the proposed method. Because of the non-label format, high selectivity and sensitivity, such a method can be a convenient and universal means for sensing various biomarkers for disease diagnosis at the early stages.

How fluorescent labels affect the kinetics of the toehold-mediated DNA strand displacement reaction

End modification of the toehold domain or near to it using fluorophore dyes or quenchers can significantly modulate the kinetics of the toehold-mediated strand displacement reaction (TMSDR) by almost two orders of magnitude.

A nonlinear neural network based on an analog DNA toehold mediated strand displacement reaction circuit

The DNA toehold mediated strand displacement reaction is one of the semi-synthetic biology technologies for next-generation computers. In this article, we present a framework for a novel nonlinear neural network based on an engineered biochemical circuit, which is constructed by several reaction modules including catalysis, degradation and adjustment reaction modules. The proposed neural network possesses an architecture that is similar to that of an error back propagation neural network, and is built of an input layer, hidden layer and output layer. As a proof of concept, we utilize this nonlinear neural network based on an analog DNA toehold mediated strand displacement reaction circuit to learn the standard quadratic form function and analyze the robustness of the nonlinear neural network toward DNA strand concentration detection, DNA strand displacement reaction rate and noise. Unlike in error back propagation neural networks, the adaptive behavior of this DNA molecular neural network system endows it with supervised learning capability. This investigation will highlight the potential of analog DNA displacement reaction circuits for implementing artificial intelligence.

Target-Activating and Toehold Displacement Ag NCs/GO Biosensor-Mediating Signal Shift and Enhancement for Simultaneous Multiple Detection.

Herein, we demonstrate that a new multicolor silver nanoclusters/graphene oxide (Ag NCs/GO) hybrid material, upon target response, undergoes a configuration transformation, based on entropy-driven enzyme-free toehold-mediated strand displacement reaction, achieving emission shift and enhancement. To realize the aim above, two different synthesis routes (route I and II) of synthesizing fluorescent Ag NCs for constructing toehold displacement Ag NCs/GO biosensor is designed and performed. Influenza A virus subtype genes (H1N1 and H5N1) as a model can efficiently initiate the operation of entropy-driven displacement reaction, resulting in activatable fluorescence. Red-emitting and green-emitting Ag NCs tethering the complementary sequence of H1N1 (pDNA1) and H5N1 (pDNA2) are indirectly immobilized on GO surface through binding with capture DNA (cDNA1 and cDNA2), respectively, forming multicolor pDNA-Ag NCs/GO nanohybrid materials. However, they do not exhibit nearly fluorescence signals attributed to energy transfer from donor Ag NCs to acceptor GO. Upon adding targets H1N1 and H5N1 (tDNA1 and tDNA2), pDNA1-Ag NCs and pDNA2-Ag NCs detach from GO, based on toehold-mediated strand displacement reaction, which interferes the energy transfer and leads to significant fluorescence enhancement. More interestingly, the activatable process is accompanied by remarkable hypsochromic shift (19 nm) or bathochromic shift (21 nm) emission with quite high fluorescence recovery rates (823.35% and 693.62%). Therefore, based on these phenomena, a novel multiple approach has been developed with the assistance of toehold displacement and Ag NCs/GO nanohybrid materials. As for the remarkable emission recovery and multichannel signal, the proposed approach displays the promising application prospect in accurate diagnosis and treatment.

Visual Test Paper for on-Site Polychlorinated Biphenyls Detection and Its Logic Gate Applications.

A visual detection method was proposed for polychlorinated biphenyls (PCBs) detection using lateral flow test paper as the sensing platform. The aptamer sequence was used to recognize the target 3,3',4,4'-tetrachlorobiphenyl (PCB77). The integration of Zn2+-dependent DNAzyme with toehold-mediated strand displacement reaction significantly improved the response signals. Gold nanoparticles were utilized as the signal tracers in the test paper, making the results visible directly by the naked eye. Under optimal conditions, the paper enables the visual detection of PCB77 as low as 10 pM without additional instrumentation. The assay displays a high selectivity for PCB77 against potential interfering molecules. The visual test paper is robust and has been applied to the detection of PCB77 in milk samples with good recovery and satisfactory accuracy. Using two different PCBs (PCB77 and PCB72) as inputs, we further fabricated OR and AND logic gates, which is conducive to the development of an intelligent detection strategy for PCBs monitoring. Given the attractive characteristics of disposability, low cost, logic operation, and intuitive output, the test paper shows great promise for on-site screening of PCBs in resource-limited areas.

Nonenzymatic DNA-Based Fluorescence Biosensor Combining Carbon Dots and Graphene Oxide with Target-Induced DNA Strand Displacement for microRNA Detection.

Based on a fluorescence “on-off-on” strategy, we fabricated a simple and highly sensitive DNA-based fluorescence biosensor for the detection of micro (mi)RNA from carbon dots (CDs) and graphene oxide (GO) without complicated and time-consuming operations. CDs were successfully synthesized and conjugated to the end of a single-stranded fuel DNA that was adsorbed onto the surface of GO through π-π stacking, resulting in fluorescence quenching. In the presence of the target miRNA let-7a, the fuel DNA was desorbed from the GO surface, and fluorescence was restored through two successive toehold-mediated strand displacement reactions on double-stranded DNA-modified gold nanoparticles. The target miRNA let-7a was recycled, leading to signal amplification. The concentration of let-7a was proportional to the degree of fluorescence recovery. Under optimal conditions, there was a good linear relationship between the relative fluorescence intensity and let-7a concentration in the range of 0.01–1 nM, with a detection limit of 7.8 pM. With its advantages of signal amplification and high biocompatibility, this fluorescence sensing strategy can be applied to the detection of a variety of target miRNAs and can guide the design of novel biosensors with improved properties.

Programming cell entry of molecules via reversible synthetic DNA circuits on cell membrane

Cellular uptake of biomolecules is crucial for regulating cell function. However, powerful and biocompatible tools for dynamically manipulating the cell entry of single-stranded DNAs (ssDNAs) remain elusive. Herein, we constructed synthetic DNA circuits on the cell membrane to program the cell entry of ssDNAs, using toehold-mediated DNA strand displacement reactions. We found that the dimerization and trimerization of cholesterol-ssDNAs enhanced membrane-anchoring and cellular uptake of ssDNAs. Moreover, we demonstrated that de-dimerization and de-trimerization of cholesterol-ssDNAs could be accomplished by inputting recovery ssDNAs into the synthetic DNA circuits, which could simultaneously decrease the cellular uptake of ssDNAs. We speculate that operating the synthetic DNA circuits on the cell membrane will be a powerful strategy for regulating the cellular uptake of exogenous materials, which has important implications for bioimaging, drug delivery, and gene therapy.

Application of the Dimeric G-Quadruplex and toehold-mediated strand displacement reaction for fluorescence biosensing of ochratoxin A

Ochratoxin A (OTA) is one of the most toxic mycotoxins that exists in various agro-products and foods. Here, a non-label and enzyme-free fluorescence biosensor for highly specific detection of OTA has been developed by the combination of toehold-mediated strand displacement reaction (TMSD) and G-quadruplex dimer/ThT (G-dimer/ThT). The DNA duplex (aptamer-IP) is composed of the anti-OTA aptamer and a single stranded initiation probe (IP). In the presence of OTA, the attachment of target to aptamer leads to the liberation of the IP, which activates the cycle TMSD amplifications of two hairpin probes (H1 and H2) accompanied by the production of numerous H1–H2 assemblies. This double-stranded H1–H2 structure results in the proximity between the 5′-end overhang tail of H1 and the 3′-end stem of H2 to liberate the pre-blocked G-dimer sequence for lighting up ThT. In addition, the method displayed a stable fluorescence emission in the high-salt media. It was successfully applied to analyze OTA in real food samples. Hence, the constructed fluorescence biosensing platform might provide a new way for OTA and other toxin analysis detection.

An activatable near-infrared fluorescent probe targeting CKIP-1 for monitoring osteoporosis in vivo

Osteoporosis (OP) is characterized by decreased bone mass and strength. Patients with OP have a higher risk of bone fragility and fracture. Currently, bone mineral density (BMD) determined by radioactive scanning is one of the most critical parameters applied in the clinical diagnosis and monitoring of therapeutic response in osteoporosis. However, radioactive scanning exposes the potential risks of long-time radiation damage. This calls for development of less toxic and highly sensitive molecular fluorescence imaging techniques. Currently, this is not possible because there are no effective indicators. Here, we found a negative correlation between osteoporosis and casein kinase 2-interacting protein-1 (CKIP-1, also known as PLEKHO1). This kinase was previously found to negatively regulate bone formation. Using this new biomarker, we designed an activatable near-infrared (NIR) fluorescent probe targeting CKIP-1 mRNA (NPC) with the toe-hold mediated strand displacement reaction and fuel amplification strategy. NIR fluorophore Cy5 and its quencher part BHQ3 were incorporated in the probe. The ratio of NPC probe and its fuel strand were also optimized.The designed detection system effectively visualized CKIP-1 at the cell level and in three different osteoporosis models. Results revealed that the NPC probe targeting CKIP-1 can be used to monitor changes in bone mass and therapy responses in vivo.

Multifunctional Clip Strand for the Regulation of DNA Strand Displacement and Construction of Complex DNA Nanodevices.

Strand displacement reactions are important bricks for the construction of various DNA nanodevices, among which the toehold-mediated strand displacement reaction is the most prevalently adopted. However, only a limited number of tools could be used to finely regulate the toehold reaction, thus restricting DNA nanodevices from being more multifunctional and powerful. Herein, we developed a regulation tool, Clip, and achieved multiple regulatory functions, including subtle adjustment of the reaction rates, allosteric strand displacement, selective activation, and resetting of the reaction. Taking advantages of the multiple functions, we constructed Clip-toehold-based DNA walking machines. They showed behaviors of controllable walking, concatenation, and programmable pathways. Furthermore, we built Clip-toehold-based AND and OR logic gates and integrated those logic gates to construct multilayer circuits, which could be reset and reused to process different input signals. We believe that the proposed Clip tool has expanded the functionality of DNA strand displacement-based nanodevices to a much more complex and diverse level and anticipate that the tool will be widely adopted in DNA nanotechnology.