DNA Strand Displacement Reactions Research Articles

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.

Programmable DNA Nanoindicator-Based Platform for Large-Scale Square Root Logic Biocomputing.

The prospect of programming molecular computing systems to realize complex autonomous tasks has advanced the design of synthetic biochemical logic circuits. One way to implement digital and analog integrated circuits is to use noncovalent hybridization and strand displacement reactions in cell-free and enzyme-free nucleic acid systems. To date, DNA-based circuits involving tens of logic gates capable of implementing basic and complex logic functions have been demonstrated experimentally. However, most of these circuits are still incapable of realizing complex mathematical operations, such as square root logic operations, which can only be carried out with 4 bit binary numbers. A high-capacity DNA biocomputing system is demonstrated through the development of a 10 bit square root logic circuit. It can calculate the square root of a 10 bit binary number (within the decimal integer 900) by designing DNA sequences and programming DNA strand displacement reactions. The input signals are optimized through the output feedback to improve performance in more complex logical operations. This study provides a more universal approach for applications in biotechnology and bioengineering.

Aptamer Displacement Reaction from Live-Cell Surfaces and Its Applications.

The DNA strand displacement reaction has had sustained scientific interest in building complicated nucleic acid-based networks. However, extending the fundamental mechanism to more diverse biomolecules in a complex environment remains challenging. Aptamers bind with targeted biomolecules with high affinity and selectivity, thus offering a promising route to link the powers of nucleic acid with diverse cues. Here, we describe three methods that allow facile and efficient displacement reaction of aptamers from the living cell surface using complement DNA (cDNA), toehold-labeled cDNA (tcDNA), and single-stranded binding protein (SSB). The kinetics of the DNA strand displacement reaction is severely affected by complex physicochemical properties of the natural membrane. Toehold-mediated and SSB-mediated aptamer displacement exhibited significantly enhanced kinetics, and they completely removed the aptamer quickly to avoid a false signal caused by aptamer internalization. Because of its simplicity, aptamer displacement enabled detection of membrane protein post-translation and improved selection efficiency of cell-SELEX.

PID and State Feedback Controllers Using DNA Strand Displacement Reactions

Nucleic acid-based chemistry is a strong candidate framework for the construction of future synthetic biomolecular control circuits. Previous work has demonstrated the capacity of circuits based on DNA strand displacement (DSD) reactions to implement digital and analogue signal processing in vivo , including in mammalian cells. To date, however, feedback control system designs attempted within this framework have been restricted to extremely simple proportional or proportional-integral controller architectures. In this letter, we significantly extend the potential complexity of such controllers by showing how time-delays, numerical differentiation (to allow PID control), and state feedback may be implemented via chemical reaction network-based designs. Our controllers are implemented and tested using VisualDSD, a rapid-prototyping tool that allows precise analysis of computational devices implemented using nucleic acids, via both deterministic and stochastic simulations of the DSD reactions.

Molecular computing for Markov chains

In this paper, it is presented a methodology for implementing arbitrarily constructed time-homogenous Markov chains with biochemical systems. Not only discrete but also continuous-time Markov chains are allowed to be computed. By employing chemical reaction networks as a programmable language, molecular concentrations serve to denote both input and output values. One reaction network is elaborately designed for each chain. The evolution of species’ concentrations over time well matches the transient solutions of the target continuous-time Markov chain, while equilibrium concentrations can indicate the steady state probabilities. Additionally, second-order Markov chains are considered for implementation, with bimolecular reactions rather than unary ones. An original scheme is put forward to compile unimolecular systems to DNA strand displacement reactions for the sake of future physical implementations. Deterministic, stochastic and DNA simulations are provided to enhance correctness, validity and feasibility.

Temporal Pattern Recognition through Analog Molecular Computation.

Living cells communicate information about physiological conditions by producing signaling molecules in a specific timed manner. Different conditions can result in the same total amount of a signaling molecule, differing only in the pattern of the molecular concentration over time. Such temporally coded information can be completely invisible to even state-of-the-art molecular sensors with high chemical specificity that respond only to the total amount of the signaling molecule. Here, we demonstrate design principles for circuits with temporal specificity, that is, molecular circuits that respond to specific temporal patterns in a molecular concentration. We consider pulsatile patterns in a molecular concentration characterized by three fundamental temporal features: time period, duty fraction, and number of pulses. We develop circuits that respond to each one of these features while being insensitive to the others. We demonstrate our design principles using general chemical reaction networks and with explicit simulations of DNA strand displacement reactions. In this way, our work develops building blocks for temporal pattern recognition through molecular computation.

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

Gold Nanoparticle Couples with Entropy-Driven Toehold-Mediated DNA Strand Displacement Reaction on Magnetic Beads: Toward Ultrasensitive Energy-Transfer-Based Photoelectrochemical Detection of miRNA-141 in Real Blood Sample.

Highly stable circulating microRNAs (miRNAs) are currently recognized as a novel potential biomarker for clinical cancer diagnosis in the early stage. However, limited by its low concentration, high sequence similarity, as well as the numerous interferences in body fluids, detection of miRNA in whole blood with sufficient selectivity and sensitivity is still challenging. Herein, we reported the integration of entropy-driven toehold-mediated DNA strand displacement (ETSD) reaction with magnetic beads (MB) toward the energy-transfer-based photoelectrochemical (PEC) detection of the prostate carcinoma (PCa) biomarker miRNA-141 in a real blood sample. In this protocol, the ETSD reaction was divided into two steps, and cooperated with magnetic separation, target extraction and amplification could be realized in a single test and ultrasensitive detection of miRNA-141 could be achieved in undiluted whole blood sample. This work proposed a new solution for sensitive biomolecular detection in a complex biological milieu and exhibited great promise for future clinical cancer diagnosis.

A facile DNA strand displacement reaction sensing strategy of electrochemical biosensor based on N-carboxymethyl chitosan/molybdenum carbide nanocomposite for microRNA-21 detection

Herein, we report a facile enzyme-free microRNA (miRNA) target-triggered strand displacement reaction (SDR) amplification strategy with ferrocene (Fc) as a signal molecule to fabricate a two-dimensional electroactive molybdenum carbide (Mo2C)-based biosensor. In the presence of miRNA-21, SDR was initiated and many hairpin DNA1 (HDNA1) and hairpin DNA2 (HDNA2) duplexes, which could be captured by probe DNA leading the Fc-modified HDNA2 close to the electrode surface, were produced continuously. MiRNA-21 could be detected by monitoring the redox signal of Fc. The prepared N-carboxymethyl chitosan/Mo2C nanocomposite featured excellent conductivity, great dispersion, and multiple functional groups (amine groups). When the nanocomposite was introduced to a miRNA biosensor electrode interface to ensure its strong connection to the DNA probe, the developed miRNA-21 biosensor demonstrated a reliable linear range of 1.0 fM to 1.0 nM with a detection limit of 0.34 fM and showed good selectivity, reproducibility, and stability. The biosensor was employed to detect miRNA-21 in human serum samples, and it showed great potential in the early clinical diagnosis of various genetic diseases.

Computing Mathematical Functions using DNA via Fractional Coding

This paper discusses the implementation of mathematical functions such as exponentials, trigonometric functions, the sigmoid function and the perceptron function with molecular reactions in general, and DNA strand displacement reactions in particular. The molecular constructs for these functions are predicated on a novel representation for input and output values: a fractional encoding, in which values are represented by the relative concentrations of two molecular types, denoted as type-1 and type-0. This representation is inspired by a technique from digital electronic design, termed stochastic logic, in which values are represented by the probability of 1’s in a stream of randomly generated 0’s and 1’s. Research in the electronic realm has shown that a variety of complex functions can be computed with remarkably simple circuitry with this stochastic approach. This paper demonstrates how stochastic electronic designs can be translated to molecular circuits. It presents molecular implementations of mathematical functions that are considerably more complex than any shown to date. All designs are validated using mass-action simulations of the chemical kinetics of DNA strand displacement reactions.

Single-Cell in Situ RNA Analysis With Switchable Fluorescent Oligonucleotides.

Comprehensive RNA analyses in individual cells in their native spatial contexts promise to transform our understanding of normal physiology and disease pathogenesis. Here we report a single-cell in situ RNA analysis approach using switchable fluorescent oligonucleotides (SFO). In this method, transcripts are first hybridized by pre-decoding oligonucleotides. These oligonucleotides subsequently recruit SFO to stain their corresponding RNA targets. After fluorescence imaging, all the SFO in the whole specimen are simultaneously removed by DNA strand displacement reactions. Through continuous cycles of target staining, fluorescence imaging, and SFO removal, a large number of different transcripts can be identified by unique fluorophore sequences and visualized at the optical resolution. To demonstrate the feasibility of this approach, we show that the hybridized SFO can be efficiently stripped by strand displacement reactions within 30 min. We also demonstrate that this SFO removal process maintains the integrity of the RNA targets and the pre-decoding oligonucleotides, and keeps them hybridized. Applying this approach, we show that transcripts can be restained in at least eight hybridization cycles with high analysis accuracy, which theoretically would enable the whole transcriptome to be quantified at the single molecule sensitivity in individual cells. This in situ RNA analysis technology will have wide applications in systems biology, molecular diagnosis, and targeted therapies.

DNA Computing Boosted by a Cationic Copolymer

AbstractThe huge information storage capability of DNA and its ability to self‐assemble can be harnessed to enable massively parallel computing in a small space. DNA‐based logic gates are designed that rely on DNA strand displacement reactions; however, computation is slow due to time‐consuming DNA reassembly processes and prone to failure as DNA is susceptible to degradation by nucleases and under certain solution conditions. Here, it is shown that the presence of a cationic copolymer boosts the speed of DNA logic gate operations that involve multiple and parallel strand displacement reactions. Two kinds of DNA molecular operations, one based on a translator gate and one on a seesaw gate, are successfully enhanced by the copolymer without tuning of computing conditions or DNA sequences. The copolymer markedly reduces operation times from hours to minutes. Moreover, the copolymer enhances nuclease resistance.

Probabilistic switching circuits in DNA

A natural feature of molecular systems is their inherent stochastic behavior. A fundamental challenge related to the programming of molecular information processing systems is to develop a circuit architecture that controls the stochastic states of individual molecular events. Here we present a systematic implementation of probabilistic switching circuits, using DNA strand displacement reactions. Exploiting the intrinsic stochasticity of molecular interactions, we developed a simple, unbiased DNA switch: An input signal strand binds to the switch and releases an output signal strand with probability one-half. Using this unbiased switch as a molecular building block, we designed DNA circuits that convert an input signal to an output signal with any desired probability. Further, this probability can be switched between 2 n different values by simply varying the presence or absence of n distinct DNA molecules. We demonstrated several DNA circuits that have multiple layers and feedback, including a circuit that converts an input strand to an output strand with eight different probabilities, controlled by the combination of three DNA molecules. These circuits combine the advantages of digital and analog computation: They allow a small number of distinct input molecules to control a diverse signal range of output molecules, while keeping the inputs robust to noise and the outputs at precise values. Moreover, arbitrarily complex circuit behaviors can be implemented with just a single type of molecular building block.

Using DNA strand displacement to control interactions in DNA-grafted colloids.

Grafting DNA oligonucleotides to colloidal particles leads to specific, reversible interactions between those particles. However, the interaction strength varies steeply and monotonically with temperature, hindering the use of DNA-mediated interactions in self-assembly. We show how the dependence on temperature can be modified in a controlled way by incorporating DNA strand-displacement reactions. The method allows us to make multicomponent systems that can self-assemble over a wide range of temperatures, invert the dependence on temperature to design colloidal systems that melt upon cooling, controllably transition between structures with different compositions, or design systems with multiple melting transitions. This wide range of behaviors can be realized simply by adding a small number of DNA strands to the solution, making the approach modular and straightforward to implement. We conclude with practical considerations for designing systems of DNA-mediated colloidal interactions.

Molecular Synthesis for Probability Theory and Stochastic Process

It is common that probability theory and stochastic process, especially Markov chains, have long been used to study and explain the behaviors of chemical reaction networks (CRNs). Nonetheless, this paper sees things from a reverse angle, devoting itself in synthesizing common probability theory and stochastic process with CRNs. The main motivation is to imitate and explore the evolution of large-scale and complex practical systems based on CRNs, by making use of the inherent parallelism and randomness. In our conference paper, a preliminary but concise approach has been put forward to synthesize the stand-alone examples such as law of total probability, Bayes’ theorem, and n-step transition of Markov chains. To make this methodology systematic and theoretically sound, we enrich and offer more solid foundation for the previous version. Rigorous stability analysis based on ordinary differential equations (ODEs) are provided. This paper further deeply discusses and distinguishes building stochastic models for CRNs and utilizing CRNs to solve stochastic problems. A joint distribution of Markov chains are implemented using molecular reactions as a showcase. In order to enhance the clearness of the results, all the simulations are done according to deterministic mass action. It is worth noting that an already mathematically proven conclusion, which states that nearly an arbitrary set of bimolecular or unimolecular reactions can be implemented by DNA strand displacement reactions, ensures the meaningfulness of our work. It is also believed that, though in its infancy, the proposed approach is also valid for other molecular synthesis than DNA, as long as the kinetics constraints are met.

Flexible regulation of DNA displacement reaction through nucleic acid-recognition enzyme and its application in keypad lock system and biosensing

Toehold-mediated DNA strand displacement reaction (SDR) plays pivotal roles for the construction of diverse dynamic DNA nanodevices. To date, many elements have been introduced into SDR system to achieve controllable activation and fine regulation. However, as the most relevant stimuli for nucleic acid involved reaction, nucleic acid-recognizing enzymes (NAEs) have received nearly no attention so far despite SDR often takes place in NAEs-enriched environment (i.e., biological fluids). Herein, we report a set of NAEs-controlled SDR strategies, which take full advantage of NAEs’ properties. In this study, three different kinds of enzymes belonging to several classes (i.e., exonuclease, endonuclease and polymerase) have been used to activate or inhibit SDR, and more importantly, some mechanisms behind these strategies on how NAEs affect SDR have also been revealed. The exploration to use NAEs as possible cues to operate SDR will expand the available toolbox to build novel stimuli-fueled DNA nanodevices and could open the door to many applications including enzyme-triggered biocomputing and biosensing.

Host–Guest Recognition-Assisted Electrochemical Release: Its Reusable Sensing Application Based on DNA Cross Configuration-Fueled Target Cycling and Strand Displacement Reaction Amplification

In this work, an elegantly designed host-guest recognition-assisted electrochemical release was established and applied in a reusable electrochemical biosensor for the detection of microRNA-182-5p (miRNA-182-5p), a prostate cancer biomarker in prostate cancer, based on the DNA cross configuration-fueled target cycling and strand displacement reaction (SDR) amplification. With such a design, the single target miRNA input could be converted to large numbers of single-stranded DNA (S1-Trp and S2-Trp) output, which could be trapped by cucurbit[8]uril methyl viologen (CB-8-MV2+) based on the host-guest recognition, significantly enhancing the sensitivity for miRNA detection. Moreover, the nucleic acids products obtained from the process of cycling amplification could be utilized sufficiently, avoiding the waste and saving the experiment cost. Impressively, by resetting a settled voltage, the proposed biosensor could release S1-Trp and S2-Trp from the electrode surface, attributing that the guest ion methyl viologen (MV2+) was reduced to MV+· under this settled voltage and formed a more-stable CB-8-MV+·-MV+· complex. Once O2 was introduced in this system, MV+· could be oxidized to MV2+, generating the complex of CB-8-MV2+ for capturing S1-Trp and S2-Trp again in only 5 min. As a result, the simple and fast regeneration of biosensor for target detection was realized on the base of electrochemical redox-driven assembly and release, overcoming the challenges of time-consuming, burdensome operations and expensive experimental cost in traditional reusable biosensors and updating the construction method for a reusable bisensor. Furthermore, the biosensor could be reused for more than 10 times with a regeneration rate of 93.20%-102.24%. After all, the conception of this work provides a novel thought for the construction of effective reusable biosensor to detect miRNA and other biomarkers and has great potential application in the area requiring the release of nucleic acids or proteins.

Programming Cell Adhesion for On-Chip Sequential Boolean Logic Functions

Programmable remodelling of cell surfaces enables high-precision regulation of cell behavior. In this work, we developed in vitro constructed DNA-based chemical reaction networks (CRNs) to program on-chip cell adhesion. We found that the RGD-functionalized DNA CRNs are entirely noninvasive when interfaced with the fluidic mosaic membrane of living cells. DNA toehold with different lengths could tunably alter the release kinetics of cells, which shows rapid release in minutes with the use of a 6-base toehold. We further demonstrated the realization of Boolean logic functions by using DNA strand displacement reactions, which include multi-input and sequential cell logic gates (AND, OR, XOR, and AND-OR). This study provides a highly generic tool for self-organization of biological systems.

Signal-on fluorescence biosensor for microRNA-21 detection based on DNA strand displacement reaction and Mg2+-dependent DNAzyme cleavage

MicroRNAs have been involved into many biological processes and are regarded as disease biomarkers. Simple, rapid, sensitive and selective method for microRNA detection is crucial for early diagnosis and therapy of diseases. In this work, sensitive fluorescence assay was developed for microRNA-21 detection based on DNA polymerase induced strand displacement amplification reaction, Mg2+-dependent DNAzyme catalysis reaction, and magnetic separation. In the presence of target microRNA-21, amounts of trigger DNA could be produced with DNA polymerase induced strand displacement amplification reaction, and the trigger DNA could be further hybridized with signal DNA, which was labeled with biotin and AMCA dye. After introduction of Mg2+, trigger DNA could form DNAzyme to cleave signal DNA. After magnetic separation, the DNA fragment with AMCA dye could give fluorescence signal, which was related to microRNA-21 concentration. Based on the two efficient signal amplifications, the developed method showed high detection sensitivity with low detection limit of 0.27fM (3σ). In addition, this fluorescence strategy also possessed excellent detection specificity, and could be applied to analyze microRNA-21 expression level in serum of cancer patient. According to the obtained results, the developed fluorescence method might be a promising detection platform for microRNA-21 quantitative analysis in biomedical research and clinical diagnosis.

DNA probes for monitoring dynamic and transient molecular encounters on live cell membranes.

Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.