Strand Displacement Technique Research Articles

Rationally Designed Dual Base Pair Mismatch Enables Toehold-Mediated Strand Displacement to Efficiently Recognize Single-Nucleotide Polymorphism without Enzymes.

The efficiency of the enzyme-free toehold-mediated strand displacement (TMSD) technique is often insufficient to detect single-nucleotide polymorphism (SNP) that possesses only single base pair mismatch discrimination. Here, we report a novel dual base pair mismatch strategy enabling TMSD biosensing for SNP detection under enzyme-free conditions when coupled with catalytic hairpin assembly (CHA) and fluorescence resonance energy transfer (FRET). The strategy is based on a competitive strand displacement reaction mechanism, affected by the thermodynamic stability originating from rationally designed dual base pair mismatch, for the specific recognition of mutant-type DNA. In particular, enzyme-free nucleic acid circuits, such as CHA, emerge as a powerful method for signal amplification. Eventually, the signal transduction of this proposed biosensor was determined by FRET between streptavidin-coated 605 nm emission quantum dots (605QDs, donor) and Cy5/biotin hybridization (acceptor, from CHA) when incubated with each other. The proposed biosensor displayed high sensitivity to the mutant target (MT) with a detection concentration down to 4.3 fM and led to high discrimination factors for all types of mismatches in multiple sequence contexts. As such, the application of this proposed biosensor to investigate mechanisms of the competitive strand displacement reaction further illustrates the versatility of our dual base pair mismatch strategy, which can be utilized for the creation of a new class of biosensors.

Finite-time regulation property of DNA feedback regulator

In dynamic DNA nanotechnology, the DNA strand displacement technique provides the cornerstone for the bottom-up design of a man-made DNA molecular system. Practically, a feedback controller for regulating the concentration of a target DNA strand to the desired level is indispensable for mediating the kinetic momentum of a molecular actuator. However, such a regulator system operates by consuming fuel strands and requires sufficient supplies of these consumables for its normal execution, indicating that, in practice, optimal controller design requires the period of time during which the regulator proceeds with normal operation to be as long as possible. The fact that the system is naturally high dimensional and nonlinear complicates the analysis of properties emerging during a finite-time period in terms of their theoretical aspects. In this paper, we first define the new concept of a “finite-time regulation property” of DNA systems in the regulation problem. Then, to theoretically analyze this regulation property, we present two-time-scale modeling based on the difference in the initial distribution of the abundance of DNA strands. Focusing on the fast mode as a subsystem with a positive quadratic structure, we propose a new method for analyzing the regulation property observed in a finite period of time.

Biochemical Logic Circuits Based on DNA Combinatorial Displacement

DNA, as an excellent nano-engineering material, contributes to a new computing model, namely, DNA computing. This model is a type of biological computing, which takes advantage of the high density and high parallelism of molecules. One of the current methods of implementing DNA computing is to construct DNA circuits, among which the toehold-mediated DNA strand displacement technique is an important method. The hybridization of toehold domains provides the start position and accelerates the branch migration process. Toehold-based DNA combinatorial displacement is a practical method for designing and implementing DNA circuits. In this paper, we designed and simulated a multiplexer using the DNA combinatorial displacement mechanism to verify its practicability. Additionally, we improved and optimized the existing logic INHIBIT gate by leveraging the DNA combinatorial displacement mechanism so that the DNA strands in the entire chemical reaction network (CRN) system are capable of coexisting in large quantities. Moreover, we applied this improvement to the demultiplexer. Our method provides more capabilities to larger and more complicated DNA integrated circuits.

Three‐input majority gate using spatially localised DNA hairpins

DNA circuits employing DNA strand displacement technique are considered as a possible replacement for silicon-based circuits, especially in implantable medical devices, because of its compatibility with bio-signals, smaller size, and light weight. In this work, a new DNA strand displacement based, three-input spatially localised majority gate using DNA hairpins is proposed. The DNA hairpins used in this majority gate are tethered to an Origami substrate, and hence, the DNA hairpins are spatially separated from each other. The localisation helps the designer to reuse the strands, without causing any spurious reactions. It is believed currently that no design is available in the literature for a localised DNA majority gate. The visual DSD software is used for simulating the proposed majority gate.

Regulation of DNA Strand Displacement Using an Allosteric DNA Toehold.

Toehold-mediated DNA strand displacement is the fundamental basis for the construction and operation of diverse DNA devices, including circuits, machines, sensors, and reconfigurable structures. Controllable activation and regulation of toeholds are critical to construct devices with multistep, autonomous, and complex behaviors. A handful of unique toehold activation mechanisms, including toehold-exchange, associative toehold, and remote toehold, have been developed and are often combined to achieve desired strand displacement behaviors and functions. Here we report an allosteric DNA toehold (A-toehold) design that allows the flexible regulation of DNA strand displacement by splitting an input strand into an A-toehold and branch migration domain. Because of its simplicity, the A-toehold mechanism can be a useful addition to the current toolbox of DNA strand displacement techniques. We demonstrated that A-toehold enabled a number of interesting functions that were previously shown using more sophisticated DNA strand displacement systems, including (1) continuously tuning the rate of strand displacement, (2) dynamic control of strand displacement reactions, and (3) selective activation of multiple strand displacement reactions. Moreover, by combining A-toehold and toehold-exchange mechanisms, we have successfully constructed a noncovalent DNA catalysis network that resembles an allosteric enzyme.

Kinetics of Proximity-Induced Intramolecular DNA Strand Displacement.

Proximity-induced intramolecular DNA strand displacement (PiDSD) is one of the key mechanisms involved in many DNA-mediated proximity assays and protein-responsive DNA devices. However, the kinetic profile of PiDSD has never been systematically examined before. Herein, we report a systematic study to explore the kinetics of PiDSD by combining the uses of three DNA strand displacement techniques, including a binding-induced DNA strand displacement to generate PiDSD, an intermolecular DNA strand-exchange strategy to measure a set of key kinetic parameters for PiDSD, and a toehold-mediated DNA strand displacement to generate fluorescence signals for the real-time monitoring of PiDSD. By using this approach, we have successfully revealed the kinetic profiles of PiDSD, determined the enhanced local effective concentrations of DNA probes that are involved in PiDSD, and identified a number of key factors that influence the kinetics of PiDSD. Our study on PiDSD establishes knowledge and strategies that can be used to guide the design and operation of various DNA-mediated proximity assays and protein-triggered DNA devices.

Synthesizing Configurable Biochemical Implementation of Linear Systems from Their Transfer Function Specifications.

The ability to engineer synthetic systems in the biochemical context is constantly being improved and has a profound societal impact. Linear system design is one of the most pervasive methods applied in control tasks, and its biochemical realization has been proposed by Oishi and Klavins and advanced further in recent years. However, several technical issues remain unsolved. Specifically, the design process is not fully automated from specification at the transfer function level, systems once designed often lack dynamic adaptivity to environmental changes, matching rate constants of reactions is not always possible, and implementation may be approximative and greatly deviate from the specifications. Building upon the work of Oishi and Klavins, this paper overcomes these issues by introducing a design flow that transforms a transfer-function specification of a linear system into a set of chemical reactions, whose input-output response precisely conforms to the specification. This system is implementable using the DNA strand displacement technique. The underlying configurability is embedded into primitive components and template modules, and thus the entire system is adaptive. Simulation of DNA strand displacement implementation confirmed the feasibility and superiority of the proposed synthesis flow.

Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.

Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA–RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.

A Novel Computational Method to Reduce Leaky Reaction in DNA Strand Displacement.

DNA strand displacement technique is widely used in DNA programming, DNA biosensors, and gene analysis. In DNA strand displacement, leaky reactions can cause DNA signals decay and detecting DNA signals fails. The mostly used method to avoid leakage is cleaning up after upstream leaky reactions, and it remains a challenge to develop reliable DNA strand displacement technique with low leakage. In this work, we address the challenge by experimentally evaluating the basic factors, including reaction time, ratio of reactants, and ion concentration to the leakage in DNA strand displacement. Specifically, fluorescent probes and a hairpin structure reporting DNA strand are designed to detect the output of DNA strand displacement, and thus can evaluate the leakage of DNA strand displacement reactions with different reaction time, ratios of reactants, and ion concentrations. From the obtained data, mathematical models for evaluating leakage are achieved by curve derivation. As a result, it is obtained that long time incubation, high concentration of fuel strand, and inappropriate amount of ion concentration can weaken leaky reactions. This contributes to a method to set proper reaction conditions to reduce leakage in DNA strand displacement.

Regulatory RNA design through evolutionary computation and strand displacement.

The discovery and study of a vast number of regulatory RNAs in all kingdoms of life over the past decades has allowed the design of new synthetic RNAs that can regulate gene expression in vivo. Riboregulators, in particular, have been used to activate or repress gene expression. However, to accelerate and scale up the design process, synthetic biologists require computer-assisted design tools, without which riboregulator engineering will remain a case-by-case design process requiring expert attention. Recently, the design of RNA circuits by evolutionary computation and adapting strand displacement techniques from nanotechnology has proven to be suited to the automated generation of DNA sequences implementing regulatory RNA systems in bacteria. Herein, we present our method to carry out such evolutionary design and how to use it to create various types of riboregulators, allowing the systematic de novo design of genetic control systems in synthetic biology.

Probabilistic reasoning with a Bayesian DNA device based on strand displacement

We present a computing model based on the DNA strand displacement technique, which performs Bayesian inference. The model will take single-stranded DNA as input data, that represents the presence or absence of a specific molecular signal (evidence). The program logic encodes the prior probability of a disease and the conditional probability of a signal given the disease affecting a set of different DNA complexes and their ratios. When the input and program molecules interact, they release a different pair of single-stranded DNA species whose ratio represents the application of Bayes' law: the conditional probability of the disease given the signal. The models presented in this paper can have the potential to enable the application of probabilistic reasoning in genetic diagnosis in vitro.

Prevalence of Neisseria Gonorrhoea among Under Graduate Female Students of University Of Port Harcpurt Using Strand Displacement and Amplification (Sda) Technique.

Background: Neisseria gonorrhoeae infection can occur asymptomatically and due to the growth requirements in the laboratory, it can be easily missed even if it is symptomatic. Isolating the organism using conventional laboratory procedures has yielded varying degrees of sensitivity and specificity. Since there is paucity of information on the prevalence rate of the organism using nucleic acid amplification technique as the detection tool, this study became pertinent. Also, the associated risk factors to the infection on female undergraduate students were highlighted. Methodology: Two hundred undergraduate, non-pregnant, asymptomatic female students who were above the age of 16 years but not above the age of 30 years were randomly selected and given questionnaires with self administrable vaginal swab sticks. The participants completed the questionnaires and provided the vaginal swab specimens which were analyzed using Strand Displacement and Amplification Technique. Results: Out of the 200 sexually active participants, 10 tested positive, giving a prevalence of 5% for Neisseria gonorrhoeae. Some of the associated risk factors elicited were; having multiple sexual partners, irregular use of condom and past history of sexually transmitted infections Conclusion: There is a need for a policy on routine screening for Neisseria gonorrhoeae since the treatment is available but the complications are danderous to reproductive health.

Isolation of a thioesterase gene from the metagenome of a mountain peak, Apharwat, in the northwestern Himalayas.

The soil metagenome of Apharwat (latitude 34.209° and longitude 74.368°) was explored for the presence of esterase encoding genes using a cultivation-independent approach, metagenomics. Among the various protocols tested, the method developed by Wechter was found to be the best for metagenome isolation from the soil under investigation. The purity of the isolated metagenomic DNA was not suitable for gene cloning. To improve the yield and purity of isolated metagenomic DNA, isothermal amplification of the isolated metagenomic DNA using phi (φ) polymerase in a strand displacement technique was performed. The amplified DNA was comparatively pure and the yield increased 50-fold. A metagenomic library was constructed in Escherichia coli (DH5α) using pUC19 as a vector with an average insert size ranging between 2 and 5 kb. Out of 10,000 clones generated, one clone carrying a ~1,870-bp insert hydrolysed tributyrin, indicating esterase activity. Sequence analysis revealed that the insert harboured three open reading frames (ORFs), of which ORF 3 encoded the esterase. Open reading frame 3 comprises 1,178 bp and encodes a putative 392 amino acid protein whose size correlates with most of the bacterial esterases. The esterase isolated in the present study is suggested to be a 4-methyl-3-oxoadipyl-CoA thioesterase (Accession No. JN717164.1), as it shows 60 % sequence similarity to the thioesterase gene of Pseudomonas reinekei (Accession No. ACZ63623.1) by BLAST, ClustalX and ClustalW analysis.

Abstractions for DNA circuit design

DNA strand displacement techniques have been used to implement a broad range of information processing devices, from logic gates, to chemical reaction networks, to architectures for universal computation. Strand displacement techniques enable computational devices to be implemented in DNA without the need for additional components, allowing computation to be programmed solely in terms of nucleotide sequences. A major challenge in the design of strand displacement devices has been to enable rapid analysis of high-level designs while also supporting detailed simulations that include known forms of interference. Another challenge has been to design devices capable of sustaining precise reaction kinetics over long periods, without relying on complex experimental equipment to continually replenish depleted species over time. In this paper, we present a programming language for designing DNA strand displacement devices, which supports progressively increasing levels of molecular detail. The language allows device designs to be programmed using a common syntax and then analysed at varying levels of detail, with or without interference, without needing to modify the program. This allows a trade-off to be made between the level of molecular detail and the computational cost of analysis. We use the language to design a buffered architecture for DNA devices, capable of maintaining precise reaction kinetics for a potentially unbounded period. We test the effectiveness of buffered gates to support long-running computation by designing a DNA strand displacement system capable of sustained oscillations.

Stepwise and reversible nanopatterning of proteins on a DNA origami scaffold

Stepwise introduction of multiple streptavidin tetramers into a nanoarray formed in a stick-like punched DNA origami as well as selective removal of predetermined tetramer from the array were successfully achieved by applying a programmed strand displacement technique to a DNA origami scaffold.

Comparison of amplification methods for transcriptomic analyses of low abundance prokaryotic RNA sources

Microarrays have established as instrumental for bacterial detection, identification, and genotyping as well as for transcriptomic studies. For gene expression analyses using limited numbers of bacteria (derived from in vivo or ex vivo origin, for example), RNA amplification is often required prior to labeling and hybridization onto microarrays. Evaluation of the fidelity of the amplification methods is crucial for the robustness and reproducibility of microarray results. We report here the first utilization of random primers and the highly processive Phi29 phage polymerase to amplify material for transcription profiling analyses. We compared two commercial amplification methods (GenomiPhi and MessageAmp kits) with direct reverse-transcription as the reference method, focusing on the robustness of mRNA quantification using either microarrays or quantitative RT-PCR. Both amplification methods using either poly-A tailing followed by in vitro transcription, or direct strand displacement polymerase, showed appreciable linearity. Strand displacement technique was particularly affordable compared to in vitro transcription-based (IVT) amplification methods and consisted in a single tube reaction leading to high amplification yields. Real-time measurements using low-, medium-, and highly expressed genes revealed that this simple method provided linear amplification with equivalent results in terms of relative messenger abundance as those obtained by conventional direct reverse-transcription.