DNA Strand Displacement Circuits Research Articles

Solution of state transfer matrix based on DNA strand displacement circuits

In recent years, DNA strand displacement (DSD) circuits have been developed in leaps and bounds. The high storage and parallelism of DNA give it an inherent advantage in the field of DNA computations. DSD is widely used in analog computations and neural network computations. However, there are few studies on solving state transfer matrix using DSD circuits. Aiming at the above problems, a scheme for solution of state transfer matrix based on DSD circuits is proposed. In this paper, the summation, subtraction, multiplication, division and exponential reaction modules are built by the DSD reactions. Based on the reaction modules, DNA chemical reaction networks of state transfer matrix are constructed. DSD circuits are built by cascading the DNA chemical reaction networks. The solution of the state transfer matrix is implemented through the DSD circuits. The Visual DSD is used to verify the practicality of DSD circuits. This scheme may provide a reference to analog computations based on DSD circuits.

Strategies to Reduce Promoter-Independent Transcription of DNA Nanostructures and Strand Displacement Complexes.

Bacteriophage RNA polymerases, in particular T7 RNA polymerase (RNAP), are well-characterized and popular enzymes for many RNA applications in biotechnology both in vitro and in cellular settings. These monomeric polymerases are relatively inexpensive and have high transcription rates and processivity to quickly produce large quantities of RNA. T7 RNAP also has high promoter-specificity on double-stranded DNA (dsDNA) such that it only initiates transcription downstream of its 17-base promoter site on dsDNA templates. However, there are many promoter-independent T7 RNAP transcription reactions involving transcription initiation in regions of single-stranded DNA (ssDNA) that have been reported and characterized. These promoter-independent transcription reactions are important to consider when using T7 RNAP transcriptional systems for DNA nanotechnology and DNA computing applications, in which ssDNA domains often stabilize, organize, and functionalize DNA nanostructures and facilitate strand displacement reactions. Here we review the existing literature on promoter-independent transcription by bacteriophage RNA polymerases with a specific focus on T7 RNAP, and provide examples of how promoter-independent reactions can disrupt the functionality of DNA strand displacement circuit components and alter the stability and functionality of DNA-based materials. We then highlight design strategies for DNA nanotechnology applications that can mitigate the effects of promoter-independent T7 RNAP transcription. The design strategies we present should have an immediate impact by increasing the rate of success of using T7 RNAP for applications in DNA nanotechnology and DNA computing.

Programming of Supercrystals Using Replicable DNA-Functionalized Colloids.

The development of self-replicating systems is of great importance in research on the origin of life. As the most iconic molecules, nucleic acids have provided prominent examples of the fabrication of self-replicating artificial nanostructures. However, it is still challenging to construct sophisticated synthetic systems that can create large-scale or three-dimensionally ordered nanomaterials using self-replicating nanostructures. By integrating a template system containing DNA-functionalized colloidal seeds with a simplified DNA strand-displacement circuit programmed subsystem to produce DNA-functionalized colloidal copies, we developed a facile enthalpy-mediated strategy to control the replication and catalytic assembly of DNA-functionalized colloids in a time-dependent manner. The replication efficiency and crystal quality of the resulting superlattice structures can be effectively increased by regulating the molar ratio of the template to the copy colloids. By constructing binary systems from two types of gold nanoparticles (or proteins), superlattice structures with different crystal symmetries can be obtained through the replication and catalytic assembly processes. This programmable enthalpy-mediated approach was easily leveraged to achieve the phase transformation and catalytic amplification of colloidal crystals starting from different initial template crystals. This work offers a potential way to construct self-replicating artificial systems that exhibit complicated phase behaviors and can produce large-scale superlattice nanomaterials.

DNA Logic Gates for Small Molecule Activation Circuits in Cells.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

Directing Uphill Strand Displacement with an Engineered Superhelicase.

The ability to finely tune reaction rates and binding energies between components has made DNA strand displacement circuits promising candidates to replicate the complex regulatory functions of biological reaction networks. However, these circuits often lack crucial properties, such as signal turnover and the ability to transiently respond to successive input signals that require the continuous input of chemical energy. Here, we introduce a method for providing such energy to strand displacement networks in a controlled fashion: an engineered DNA helicase, Rep-X, that transiently dehybridizes specific DNA complexes, enabling the strands in the complex to participate in downstream hybridization or strand displacement reactions. We demonstrate how this process can direct the formation of specific metastable structures by design and that this dehybridization process can be controlled by DNA strand displacement reactions that effectively protect and deprotect a double-stranded complex from unwinding by Rep-X. These findings can guide the design of active DNA strand displacement regulatory networks, in which sustained dynamical behavior is fueled by helicase-regulated unwinding.

Temporal logic circuits implementation using a dual cross-inhibition mechanism based on DNA strand displacement.

Molecular circuits crafted from DNA molecules harness the inherent programmability and biocompatibility of DNA to intelligently steer molecular machines in the execution of microscopic tasks. In comparison to combinational circuits, DNA-based temporal circuits boast supplementary capabilities, allowing them to proficiently handle the omnipresent temporal information within biochemical systems and life sciences. However, the lack of temporal mechanisms and components proficient in comprehending and processing temporal information presents challenges in advancing DNA circuits that excel in complex tasks requiring temporal control and time perception. In this study, we engineered temporal logic circuits through the design and implementation of a dual cross-inhibition mechanism, which enables the acceptance and processing of temporal information, serving as a fundamental building block for constructing temporal circuits. By incorporating the dual cross-inhibition mechanism, the temporal logic gates are endowed with cascading capabilities, significantly enhancing the inhibitory effect compared to a cross-inhibitor. Furthermore, we have introduced the annihilation mechanism into the circuit to further augment the inhibition effect. As a result, the circuit demonstrates sensitive time response characteristics, leading to a fundamental improvement in circuit performance. This architecture provides a means to efficiently process temporal signals in DNA strand displacement circuits. We anticipate that our findings will contribute to the design of complex temporal logic circuits and the advancement of molecular programming.

Effects of fluorescent pair on the kinetics of DNA strand displacement reaction

Fluorescent labels are widely used in the characterizations of DNA-based reaction network operations. We systematically studied the effects of commonly used fluorescent pairs on thermal stabilities of signal-substrate duplex and the strand displacement kinetics. It is demonstrated that the modifications of duplex with fluorescent pairs stabilize DNA duplex by up to 3.5 °C, and the kinetics of DNA strand displacement circuit is also evidently slowed down. These results highlight the importance of fluorescent pairs towards the kinetic modulation in designing nucleic acid probes and complex DNA dynamic circuits.

Solution of Simultaneous Higher Order Equations Based on DNA Strand Displacement Circuit.

Currently, DNA strand displacement is often used to build neural networks or solve logical problems. While there are few studies on the use of DNA strand displacement to solve the higher order equations. In this paper, the catalysis, degradation, annihilation and adjusted reaction modules are built through DNA strand displacement. The chemical reaction networks of the corresponding higher order equations and simultaneous equations are established through these modules, and these chemical reaction networks can be used to build analog circuits to solve binary primary simultaneous equations and binary quadratic simultaneous equations. Finally, through Visual DSD software verification, this design can realize the solution of binary primary simultaneous equations and binary quadratic simultaneous equations, which provides a reference for DNA computation in the future.

DNA Strand-Displacement Temporal Logic Circuits.

Molecular circuits capable of processing temporal information are essential for complex decision making in response to both the presence and history of a molecular environment. A particular type of temporal information that has been recognized to be important is the relative timing of signals. Here we demonstrate the strategy of temporal memory combined with logic computation in DNA strand-displacement circuits capable of making decisions based on specific combinations of inputs as well as their relative timing. The circuit encodes the timing information on inputs in a set of memory strands, which allows for the construction of logic gates that act on current and historical signals. We show that mismatches can be employed to reduce the complexity of circuit design and that shortening specific toeholds can be useful for improving the robustness of circuit behavior. We also show that a detailed model can provide critical insights for guiding certain aspects of experimental investigations that an abstract model cannot. We envision that the design principles explored in this study can be generalized to more complex temporal logic circuits and incorporated into other types of circuit architectures, including DNA-based neural networks, enabling the implementation of timing-dependent learning rules and opening up new opportunities for embedding intelligent behaviors into artificial molecular machines.

Exponential Function Computation Based on DNA Strand Displacement Circuits

Due to its high programmability and storage, DNA circuits have been widely used in biological computing. In this paper, the addition, subtraction, multiplication, division, n-order and 1/n-order gates are built through DNA strand displacement reactions. The chemical reaction networks of the exponential function are established by using the six DNA analog computation gates. The integrated DNA strand displacement circuits are built through the chemical reaction networks. The exponential function polynomials can be computed through the integrated DNA strand displacement circuits. Finally, through visual DSD software verification, this design can realise the computation of exponential function polynomials, which provides a reference for solving exponential function equations and neural network computations in the future.

Encryption Algorithm Based on DNA Strand Displacement and DNA Sequence Operation.

DNA strand displacement is introduced in this study and used to construct an analog DNA strand displacement chaotic system based on six reaction modules in nanoscale size. The DNA strand displacement circuit is employed in encryption as a chaotic generator to produce chaotic sequences. In the encryption algorithm, we convert chaotic sequences to binary ones by comparing the concentration of signal DNA strand. Simulation results show that the encryption scheme is sensitive to the keys, and key space is large enough to resist the brute-force attacks, furthermore algorithm has a high capacity to resist statistic attack. Based on robustness analysis, our proposed encryption scheme is robust to the DNA strand displacement reaction rate control, noise and concentration detection to a certain extent.

A Novel Adaptive Linear Neuron Based on DNA Strand Displacement Reaction Network.

Analog DNA strand displacement circuits can be used to build artificial neural network due to the continuity of dynamic behavior. In this study, DNA implementations of novel catalysis, novel degradation and adjustment reaction modules are designed and used to build an analog DNA strand displacement reaction network. A novel adaptive linear neuron (ADALINE) is constructed by the ordinary differential equations of an ideal formal chemical reaction network, which is built by reaction modules. When reaction network approaches equilibrium, the weights of the ADALINE are updated without learning algorithm. Simulation results indicate that, ADALINE based on the analog DNA strand displacement circuit has ability to implement the learning function of the ADALINE based on the ideal formal chemical reaction networks, and fit a class of linear function.

Direct Comparison of d-DNA and l-DNA Strand-Displacement Reactions in Living Mammalian Cells.

To overcome technical challenges associated with the use of DNA strand-displacement circuits in vivo, including degradation by cellular nucleases, researchers are increasingly turning to bio-orthogonal l-DNA. Although enhanced stability and improved performance of l-DNA-based circuits within living cells are often implied, direct experimental evidence has not been provided. Herein, we directly compare the functional stability and kinetics of d-DNA and l-DNA strand-displacement in live cells for the first time. We show that l-DNA strand-displacement reaction systems have minimal "leak", fast reaction kinetics, and prolonged stability inside living cells as compared to conventional d-DNA. Furthermore, using "heterochiral" strand-displacement, we demonstrate that biostable l-DNA reaction components can be easily interfaced with native DNA inside cells. Overall, our results strongly support the broader adoption of l-DNA in the field of DNA molecular circuitry, especially for in vivo applications.

Verifying polymer reaction networks using bisimulation

The Chemical Reaction Network model has been proposed as a programming language for molecular programming. Methods to implement arbitrary CRNs using DNA strand displacement circuits have been investigated, as have methods to prove the correctness of those or other implementations. However, the stochastic Chemical Reaction Network model is provably not deterministically Turing-universal, that is, it is impossible to create a stochastic CRN where a given output molecule is produced if and only if an arbitrary Turing machine accepts. A DNA stack machine that can simulate arbitrary Turing machines with minimal slowdown deterministically has been proposed, but it uses unbounded polymers that cannot be modeled as a Chemical Reaction Network. We propose an extended version of a Chemical Reaction Network that models unbounded linear polymers made from a finite number of monomers. This Polymer Reaction Network model covers the DNA stack machine, as well as copy-tolerant Turing machines and some examples from biochemistry. We adapt the bisimulation method of verifying DNA implementations of Chemical Reaction Networks to our model, and use it to prove the correctness of the DNA stack machine implementation. We define a subclass of single-locus Polymer Reaction Networks and show that any member of that class can be bisimulated by a network using only four primitives, suggesting a method of DNA implementation. Finally, we prove that deciding whether an implementation is a bisimulation is Π20-complete, and thus undecidable in the general case, although it is tractable in many special cases of interest. We hope that the ability to model and verify implementations of Polymer Reaction Networks will aid in the rational design of molecular systems.

Programming colloidal bonding using DNA strand-displacement circuitry

As a strategy for regulating entropy, thermal annealing is a commonly adopted approach for controlling dynamic pathways in colloid assembly. By coupling DNA strand-displacement circuits with DNA-functionalized colloid assembly, we developed an enthalpy-mediated strategy for achieving the same goal while working at a constant temperature. Using this tractable approach allows colloidal bonding to be programmed for synchronization with colloid assembly, thereby realizing the optimal programmability of DNA-functionalized colloids. We applied this strategy to conditionally activate colloid assembly and dynamically switch colloid identities by reconfiguring DNA molecular architectures, thereby achieving orderly structural transformations; leveraging the advantage of room-temperature assembly, we used this method to prepare a lattice of temperature-sensitive proteins and gold nanoparticles. This approach bridges two subfields: dynamic DNA nanotechnology and DNA-functionalized colloid programming.

Light-Controlled, Toehold-Mediated Logic Circuit for Assembly of DNA Tiles.

Inspired by cytoskeletal structures that respond sensitively to environmental changes and chemical inputs, we report a strategy to trigger and finely control the assembly of stimulus-responsive DNA nanostructures with light under isothermal conditions. The strategy is achieved via integrating an upstream light-controlled, toehold-mediated DNA strand displacement circuit with a downstream DNA tile self-assembly process. By rationally designing an upstream DNA strand module, we further transform the upstream DNA strand displacement circuit to an "AND gate" circuit to control the assembly of DNA nanostructures. This example represents the demonstration of the spatial and temporal assembly of DNA nanostructures using a noninvasive chemical input. Such a light-controlled DNA logic circuit not only adds a new element to the tool box of DNA nanotechnology but also inspires us to assemble complex and responsive nanostructures.

DNA-based communication in populations of synthetic protocells.

Developing molecular communication platforms based on orthogonal communication channels is a crucial step towards engineering artificial multicellular systems. Here, we present a general and scalable platform entitled 'Biomolecular Implementation of Protocellular Communication' (BIO-PC) to engineer distributed multichannel molecular communication between populations of non-lipid semipermeable microcapsules. Our method leverages the modularity and scalability of enzyme-free DNA strand-displacement circuits to develop protocellular consortia that can sense, process and respond to DNA-based messages. We engineer a rich variety of biochemical communication devices capable of cascaded amplification, bidirectional communication and distributed computational operations. Encapsulating DNA strand-displacement circuits further allows their use in concentrated serum where non-compartmentalized DNA circuits cannot operate. BIO-PC enables reliable execution of distributed DNA-based molecular programs in biologically relevant environments and opens new directions in DNA computing and minimal cell technology.

Improving the Performance of DNA Strand Displacement Circuits by Shadow Cancellation.

DNA strand displacement circuits are powerful tools that can be rationally engineered to implement molecular computing tasks because they are programmable, cheap, robust, and predictable. A key feature of these circuits is the use of catalytic gates to amplify signal. Catalytic gates tend to leak; that is, they generate output signal even in the absence of intended input. Leaks are harmful to the performance and correct operation of DNA strand displacement circuits. Here, we present "shadow cancellation", a general-purpose technique to mitigate leak in catalytic DNA strand displacement circuits. Shadow cancellation involves constructing a parallel shadow circuit that mimics the primary circuit and has the same leak characteristics. It is situated in the same test tube as the primary circuit and produces "anti-background" DNA strands that cancel "background" DNA strands produced by leak. We demonstrate the feasibility and strength of the shadow leak cancellation approach through a challenging test case, a cross-catalytic feedback DNA amplifier circuit that leaks prodigiously. Shadow cancellation dramatically reduced the leak of this circuit and improved the signal-to-background difference by several fold. Unlike existing techniques, it makes no modifications to the underlying amplifier circuit and is agnostic to its leak mechanism. Shadow cancellation also showed good robustness to concentration errors in multiple scenarios. This work introduces a direction in leak reduction techniques for DNA strand displacement amplifier circuits and can potentially be extended to other molecular amplifiers.

Kinetic Trans-Assembly of DNA Nanostructures.

The central dogma of molecular biology is the principal framework for understanding how nucleic acid information is propagated and used by living systems to create complex biomolecules. Here, by integrating the structural and dynamic paradigms of DNA nanotechnology, we present a rationally designed synthetic platform that functions in an analogous manner to create complex DNA nanostructures. Starting from one type of DNA nanostructure, DNA strand displacement circuits were designed to interact and pass along the information encoded in the initial structure to mediate the self-assembly of a different type of structure, the final output structure depending on the type of circuit triggered. Using this concept of a DNA structure "trans-assembling" a different DNA structure through nonlocal strand displacement circuitry, four different schemes were implemented. Specifically, 1D ladder and 2D double-crossover (DX) lattices were designed to kinetically trigger DNA circuits to activate polymerization of either ring structures or another type of DX lattice under enzyme-free, isothermal conditions. In each scheme, the desired multilayer reaction pathway was activated, among multiple possible pathways, ultimately leading to the downstream self-assembly of the correct output structure.

Design and Analysis of Compact DNA Strand Displacement Circuits for Analog Computation Using Autocatalytic Amplifiers.

A main goal in DNA computing is to build DNA circuits to compute designated functions using a minimal number of DNA strands. Here, we propose a novel architecture to build compact DNA strand displacement circuits to compute a broad scope of functions in an analog fashion. A circuit by this architecture is composed of three autocatalytic amplifiers, and the amplifiers interact to perform computation. We show DNA circuits to compute functions sqrt(x), ln(x) and exp(x) for x in tunable ranges with simulation results. A key innovation in our architecture, inspired by Napier's use of logarithm transforms to compute square roots on a slide rule, is to make use of autocatalytic amplifiers to do logarithmic and exponential transforms in concentration and time. In particular, we convert from the input that is encoded by the initial concentration of the input DNA strand, to time, and then back again to the output encoded by the concentration of the output DNA strand at equilibrium. This combined use of strand-concentration and time encoding of computational values may have impact on other forms of molecular computation.