Toehold-mediated Strand Displacement Reaction Research Articles

DNA logic gate based on metallo-toehold strand displacement.

DNA is increasingly being used as an ideal material for the construction of nanoscale structures, circuits, and machines. Toehold-mediated DNA strand displacement reactions play a very important role in these enzyme-free constructions. In this study, the concept of metallo-toehold was utilized to further develop a mechanism for strand displacement driven by Ag+ ions, in which the intercalation of cytosine–cytosine mismatched base pairs on the toeholds provides additional control by varying of the concentration of Ag+ ions. The characteristics of displacement reaction in response to different concentration of Ag+ ions are investigated by fluorescence spectral and non-denaturing polyacrylamide gel electrophoresis. The reaction can successfully occur when the concentration of Ag+ ions is suitabe; excess Ag+ ions block the reaction. Furthermore, the displacement reaction can be tuned and controlled most efficiently under the condition of two C:C mismatched base pairs placed on the six-nt toehold. Based on our research, a mechanism was developed to construct Boolean logic gate AND and OR by employing strand displacement reaction as a tool, Ag+ and Hg2+ as input.

Yield Improving Strategy for SNP Detection on DNA Origami Chip by Employing Au Nanoparticles

Detection of single nucleotide polymorphisms (SNPs) is critical for early stage diagnosis. However, available clinical methods for detecting SNPs require strict assay conditions or complicated enzyme reactions. DNA hybridization by Au nanoparticles is a new microscopic method for DNA detection, which offers many advantages over traditional ones. But currently, this method is limitedly used because of low yield of hybridization. Here we report novel strategies which use toehold-mediated strand displacement on DNA origami Chip by employing AuNPs, and we greatly improved the yield of hybridization to 80% from less than 10%. Based on the kinetic characteristic of toehold-mediated strand displacement reaction, DNA strands with unmatched sequences can be easily replaced by fully complementary ones, and the exchange rate depends on the length of the toehold region. Therefore, variation on ssDNA sequence can be differentiated by their different displacement time. With this approach we detect SNPs of age-related macular degeneration on DNA origami chip. This approach offers a new, simple and sensitive way to detect SNP. Keywords: Age-related macular degeneration (AMD), atomic force microscope (AFM), Au nanoparticle (AuNP), DNA origami chips, single- nucleotide polymorphisms (SNPs).

Ultraselective electrochemiluminescence biosensor based on locked nucleic acid modified toehold-mediated strand displacement reaction and junction-probe

Locked nucleic acid (LNA) is applied in toehold-mediated strand displacement reaction (TMSDR) to develop a junction-probe electrochemiluminescence (ECL) biosensor for single-nucleotide polymorphism (SNP) detection in the BRCA1 gene related to breast cancer. More than 65-fold signal difference can be observed with perfectly matched target sequence to single-base mismatched sequence under the same conditions, indicating good selectivity of the ECL biosensor.

Logic nanoparticle beacon triggered by the binding-induced effect of multiple inputs.

Recently, the toehold-mediated DNA strand displacement reaction has been widely used in detecting molecular signals. However, traditional strand displacement, without cooperative signaling among DNA inputs, is insufficient for the design of more complicated nanodevices. In this work, a logic computing system is established using the cooperative "binding-induced" mechanism, based on the AuNP-based beacons, in which five kinds of multiple-input logic gates have been constructed. This system can recognize DNA and protein streptavidin simultaneously. Finally, the manipulations of the logic system are also demonstrated by controlling programmed conjugate DNA/AuNP clusters. This study provides the possibility of detecting multiple input signals and designing complex nanodevices that can be potentially applied to the detection of multiple molecular targets and the construction of large-scale DNA-based computation.

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples.

Catalytic Molecular Logic Devices by DNAzyme Displacement

Chemical reactions catalyzed by DNAzymes offer a route to programmable modification of biomolecules for therapeutic purposes. To this end, we have developed a new type of catalytic DNA-based logic gates in which DNAzyme catalysis is controlled via toehold-mediated strand displacement reactions. We refer to these as DNAzyme displacement gates. The use of toeholds to guide input binding provides a favorable pathway for input recognition, and the innate catalytic activity of DNAzymes allows amplification of nanomolar input concentrations. We demonstrate detection of arbitrary input sequences by rational introduction of mismatched bases into inhibitor strands. Furthermore, we illustrate the applicability of DNAzyme displacement to compute logic functions involving multiple logic gates. This work will enable sophisticated logical control of a range of biochemical modifications, with applications in pathogen detection and autonomous theranostics.

Disposable Strip Biosensor for Visual Detection of Hg2+ Based on Hg2+-Triggered Toehold Binding and Exonuclease III-Assisted Signal Amplification

A disposable strip biosensor for the visual detection of Hg(2+) in aqueous solution has been constructed on the basis of Hg(2+)-triggered toehold binding and exonuclease III (Exo III)-assisted signal amplification. Thymine-thymine (T-T) mismatches in the toehold domains can serve as specific recognition elements for Hg(2+) binding with the help of T-Hg(2+)-T base pairs to initiate toehold-mediated strand displacement reaction. Exo III-catalyzed target recycling strategy is introduced to improve the sensitivity. Using gold nanoparticles as a tracer, the output signals can be directly observed by the naked eye. The assay is ultrasensitive, enabling the visual detection of trace amounts of Hg(2+) as low as 1 pM without instrumentation. This sensing system also displays remarkable specificity to Hg(2+) against other possible competing ions. This sensor is robust and can be applied to the reliable monitoring of spiked Hg(2+) in environmental water samples with good recovery and accuracy. With the advantages of cost-effectiveness, simplicity, portability, and convenience, the disposable strip biosensor will be a promising candidate for point-of-use monitoring of Hg(2+) in environmental and biological samples.

Detection of Single-Nucleotide Polymorphisms Using an ON–OFF Switching of Regenerated Biosensor Based on a Locked Nucleic Acid-Integrated and Toehold-Mediated Strand Displacement Reaction

Although various strategies have been reported for single-nucleotide polymorphisms (SNPs) detection, development of a time-saving, specific, and regenerated electrochemical sensing platform still remains a realistic goal. In this study, an ON-OFF switching of a regenerated biosensor based on a locked nucleic acid (LNA)-integrated and toehold-mediated strand displacement reaction technique is constructed for detection of SNPs. The LNA-integrated and methylene blue-labeled capture probe with an external toehold is designed to switch on the sensing system. The mutant-type DNA probe completes complementary with the capture probe to trigger the strand displacement reaction, which switches off the sensing system. However, when the single-base mismatched wild-type DNA probe is presented, the strand displacement reaction cannot be achieved; therefore, the sensing system still keeps the ON state. This DNA sensor is stable over five reuses. We further testify that the LNA-integrated sequence has better recognition ability for SNPs detection compared to the DNA-integrated sequence. Moreover, this DNA senor exhibits a remarkable discrimination capability of SNPs among abundant wild-type targets and 6000-fold (m/m) excess of genomic DNA. In addition, it is selective enough in complex and contaminant-ridden samples, such as human urine, soil, saliva, and beer. Overall, these results demonstrate that this reliable DNA sensor is easy to be fabricated, simple to operate, and stable enough to be readily regenerated.

DNA “Nano-Claw”: Logic-Based Autonomous Cancer Targeting and Therapy

Cell types, both healthy and diseased, can be classified by inventories of their cell-surface markers. Programmable analysis of multiple markers would enable clinicians to develop a comprehensive disease profile, leading to more accurate diagnosis and intervention. As a first step to accomplish this, we have designed a DNA-based device, called "Nano-Claw". Combining the special structure-switching properties of DNA aptamers with toehold-mediated strand displacement reactions, this claw is capable of performing autonomous logic-based analysis of multiple cancer cell-surface markers and, in response, producing a diagnostic signal and/or targeted photodynamic therapy. We anticipate that this design can be widely applied in facilitating basic biomedical research, accurate disease diagnosis, and effective therapy.

Sequence selective capture, release and analysis of DNA using a magnetic microbead-assisted toehold-mediated DNA strand displacement reaction.

This paper reports on the modification of magnetic beads with oligonucleotide capture probes with a specially designed pendant toehold (overhang) aimed specifically to capture double-stranded PCR products. After capture, the PCR products were selectively released from the magnetic beads by means of a toehold-mediated strand displacement reaction using short artificial oligonucleotide triggers and analysed using capillary electrophoresis. The approach was successfully shown on two genes widely used in human DNA genotyping, namely human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) and amelogenin.

DNA Tetraplexes-Based Toehold Activation for Controllable DNA Strand Displacement Reactions

Most of the dynamic DNA devices are rationally constructed by utilizing toehold-mediated DNA strand displacement reactions. However, such approaches have been mainly limited to the operation with double-stranded hybridization and lack the versatility of DNA scaffold responses for additional levels of controlling DNA strand displacement reactions. Herein, we propose a toehold activation strategy based on the DNA tetraplex (G-quadruplex or i-motif), where the toehold domain is designed by attaching a complementary single-stranded segment (CS) to a G-rich/C-rich segment. Modulating G-quartet/C·C(+) numbers and/or the CS lengths can easily tune the strand displacement kinetics. This scheme allows fine control of DNA strand displacement rates over 2 orders of magnitude by adjusting the concentration of various environmental stimuli. This strategy expands the rule set of designing dynamic DNA devices and will be useful in building diverse environmental stimuli-fuelled molecular devices.

A reusable quartz crystal microbalance biosensor for highly specific detection of single-base DNA mutation

A reusable quartz crystal microbalance (QCM) biosensor based on the toehold-mediated strand displacement reaction (SDR) is first developed for highly specific detection of single-base DNA mutation. N5, N10-methylenetetrahydrofolate reductase (MTHFR) gene C677T mutation is chosen as a model to investigate the performance of the constructed biosensor. The capture DNA immobilized on the gold electrode surface of QCM hybridizes with the conjugate of biotinylated reporter DNA and streptavidin (abbreviated to reporter probe) to form the recognition layer. A toehold domain in the reporter DNA is specifically designed to bind the complementary mutant DNA and trigger the SDR, eventually resulting in the release of the complex of reporter probe and mutant DNA from the gold surface, and then the recognition layer is mildly regenerated by subsequently injecting the reporter probe again. The fabricated biosensor shows good repeatability for the mutant DNA, as more than 87% signal intensity of the sensor remained after six repetitive measurements. A linear range of 1–75nM is achieved with a detection limit of 0.8nM at room temperature. This biosensor also affords remarkable specificity to the mutant DNA against its wild-type DNA with a discrimination factor of 33. The potential of applying this biosensor in screening real samples is confirmed by spiked HeLa cells lysate with a recovery of 108%. The proposed sensing strategy provides a simple and universal solution for repetitive and highly specific detection of single-base DNA mutation.

Toehold-Mediated Nonenzymatic DNA Strand Displacement As a Platform for DNA Genotyping

Toehold-mediated DNA strand displacement provides unique advantages in the construction and manipulation of multidimensional DNA nanostructures as well as nucleic acid sequence analysis. We demonstrate a step change in the use of toehold-mediated DNA strand displacement reactions, where a double-stranded DNA duplex, containing a single-stranded toehold domain, enzymatically generated and then treated as a molecular target for analysis. The approach was successfully implemented for human DNA genotyping, such as gender identification where the amelogenin gene was used as a model target system, and detecting single nucleotide polymorphisms of human mitochondrial DNA. Kinetics of the strand displacement was monitored by the quenched Förster resonance energy transfer effect.

Enzyme‐Free Unlabeled DNA Logic Circuits Based on Toehold‐Mediated Strand Displacement and Split G‐Quadruplex Enhanced Fluorescence

Adopting fluorescence of PPIX enhanced by a split G-quadruplex and toehold mediated strand displacement reaction, a series of unlabeled fluorescent logic gates is set up and some of them are cascaded into circuits. Controlled release of PPIX, which is also a photosensitizer in photodynamic diagnosis and therapy, is realized by this circuit, making it a wise choice for DNA computing.

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction

We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection.

Highly Selective Detection of Single-Nucleotide Polymorphisms Using a Quartz Crystal Microbalance Biosensor Based on the Toehold-Mediated Strand Displacement Reaction

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.

Synchronized Assembly of Gold Nanoparticles Driven by a Dynamic DNA-Fueled Molecular Machine

A strategy for gold nanoparticle (AuNP) assembly driven by a dynamic DNA-fueled molecular machine is revealed here. In this machine, the aggregation of DNA-functionalized AuNPs is regulated by a series of toehold-mediated strand displacement reactions of DNA. The aggregation rate of the AuNPs can be regulated by controlling the amount of oligonucleotide catalyst. The versatility of the dynamic DNA-fueled molecular machine in the construction of two-component "OR" and "AND" logic gates has been demonstrated. This newly established strategy may find broad potential applications in terms of building up an "interface" that allows the combination of the strand displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, ultimately leading to the fabrication of a wide range of complex multicomponent devices and architectures.

Meta-DNA: synthetic biology via DNA nanostructures and hybridization reactions.

Can a wide range of complex biochemical behaviour arise from repeated applications of a highly reduced class of interactions? In particular, can the range of DNA manipulations achieved by protein enzymes be simulated via simple DNA hybridization chemistry? In this work, we develop a biochemical system which we call meta-DNA (abbreviated as mDNA), based on strands of DNA as the only component molecules. Various enzymatic manipulations of these mDNA molecules are simulated via toehold-mediated DNA strand displacement reactions. We provide a formal model to describe the required properties and operations of our mDNA, and show that our proposed DNA nanostructures and hybridization reactions provide these properties and functionality. Our meta-nucleotides are designed to form flexible linear assemblies (single-stranded mDNA (ssmDNA)) analogous to single-stranded DNA. We describe various isothermal hybridization reactions that manipulate our mDNA in powerful ways analogous to DNA-DNA reactions and the action of various enzymes on DNA. These operations on mDNA include (i) hybridization of ssmDNA into a double-stranded mDNA (dsmDNA) and heat denaturation of a dsmDNA into its component ssmDNA, (ii) strand displacement of one ssmDNA by another, (iii) restriction cuts on the backbones of ssmDNA and dsmDNA, (iv) polymerization reactions that extend ssmDNA on a template to form a complete dsmDNA, (v) synthesis of mDNA sequences via mDNA polymerase chain reaction, (vi) isothermal denaturation of a dsmDNA into its component ssmDNA, and (vii) an isothermal replicator reaction that exponentially amplifies ssmDNA strands and may be modified to allow for mutations.