DNA Strand Displacement Research Articles

Implementation of an Ultrasensitive Biomolecular Controller for Enzymatic Reaction Processes With Delay Using DNA Strand Displacement.

In this article, a set of abstract chemical reactions has been employed to construct a novel nonlinear biomolecular controller, i.e, the Brink controller (BC) with direct positive autoregulation (DPAR) (namely BC-DPAR controller). In comparison to dual rail representation-based controllers such as the quasi sliding mode (QSM) controller, the BC-DPAR controller directly reduces the number of CRNs required for realizing an ultrasensitive input-output response because it does not involve the subtraction module, reducing the complexity of DNA implementations. Then, the action mechanism and steady-state condition constraints of two nonlinear controllers, BC-DPAR controller and QSM controller, are investigated further. Considering the mapping relationship between CRNs and DNA implementation, a CRNs-based enzymatic reaction process with delay is constructed, and a DNA strand displacement (DSD) scheme representing time delay is proposed. The BC-DPAR controller, when compared to the QSM controller, can reduce the number of abstract chemical reactions and DSD reactions required by 33.3% and 31.8%, respectively. Finally, an enzymatic reaction scheme with BC-DPAR controller is designed using DSD reactions. According to the findings, the enzymatic reaction process's output substance can approach the target level at a quasi-steady state in both delay-free and non-zero delay conditions, but the target level can only be achieved during a finite-time period, mainly due to the fuel stand depletion.

Supervised Learning in a Multilayer, Nonlinear Chemical Neural Network.

The development of programmable or trainable molecular circuits is an important goal in the field of molecular programming. Multilayer, nonlinear, artificial neural networks are a powerful framework for implementing such functionality in a molecular system, as they are provably universal function approximators. Here, we present a design for multilayer chemical neural networks with a nonlinear hyperbolic tangent transfer function. We use a weight perturbation algorithm to train the neural network which uses a simple construction to directly approximate the loss derivatives required for training. We demonstrate the training of this system to learn all 16 two-input binary functions from a common starting point. This work thus introduces new capabilities in the field of adaptive and trainable chemical reaction network (CRN) design. It also opens the door to potential future experimental implementations, including DNA strand displacement reactions.

DNA strand displacement and TdT-Mediated DNA extension for swift, convenient, and quantitative evaluation of sperm DNA integrity and its clinical implications

DNA integrity is crucial for the clinical pregnancy outcome and offspring health, while detection methods currently used (comet assay, TUNNEL assay, SCSA, etc.) can only provide the ratio of positive sperms at the cellular level and are unable to quantitatively detect the breakpoints at the DNA molecular level. Herein, we developed a detection system based on terminal deoxynucleotidyl transferase and DNA strand displacement fluorescent probe, which could efficiently and conveniently measure the number of 3′-OH (equivalent to the number of breakpoints). We further investigated the use of this technique in assisted reproduction after completing the principle verification, system optimization, and research on analytical performance. The detection system was shown to have a good linear range from 0.01 nM to 4 nM, using single-stranded DNA with 3′-OH end as the calibrator. The system underwent thorough optimization for stability and accuracy. In comparison to the widely accepted index DFI detected by SCSA, the new system demonstrated reasonable correlation and better prediction efficiency. Its applicability was also proven through its use in assisted reproductive technology procedures.

Protocell Communication through the Eyes of Synthetic Organic Chemists

AbstractThe bottom‐up fabrication of synthetic cells (protocells) from molecules and materials, is a major challenge of modern chemistry. A significant breakthrough has been the engineering of protocells capable of chemical communication using bio‐derived molecules and ex situ stabilised cell machineries. These, however, suffer from short shelf‐lives, high costs, and require mild aqueous conditions. In this Concept Article we analyse the chemistry at the heart of protocell communication to highlight new opportunities for synthetic chemists in protocell engineering. Specifically, we (i) categorise the main bio‐derived chemical communication machineries in enzyme cascades, DNA strand displacement, and gene‐mediated communication; (ii) review the chemistries of these signal transduction machineries; and (iii) introduce new types of bio‐inspired, fully synthetic artificial enzymes to replace their natural counterparts. Developing protocells that incorporate synthetic analogues of bio‐derived signal transduction machineries will improve the robustness, stability, and versatility of protocells, and broaden their applications to highly strategic fields such as photocatalysis and fine chemicals production.

Application of recombinant human pyruvate kinase in recombinase polymerase amplification

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the invitro synthesized urease subunit β (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20ng/μL and 10mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use.

Timed Pulses in DNA Strand Displacement Reactions.

Inspired by naturally occurring regulatory mechanisms that allow complex temporal pulse features with programmable delays, we demonstrate here a strategy to achieve temporally programmed pulse output signals in DNA-based strand displacement reactions (SDRs). To achieve this, we rationally designed input strands that, once bound to their target duplex, can be gradually degraded, resulting in a pulse output signal. We also designed blocker strands that suppress strand displacement and determine the time at which the pulse reaction is generated. We show that by controlling the degradation rate of blocker and input strands, we can finely control the delayed pulse output over a range of 10 h. We also prove that it is possible to orthogonally delay two different pulse reactions in the same solution by taking advantage of the specificity of the degradation reactions for the input and blocker strands. Finally, we show here two possible applications of such delayed pulse SDRs: the time-programmed pulse decoration of DNA nanostructures and the sequentially appearing and self-erasing formation of DNA-based patterns.

Three-input logic gate based on DNA strand displacement reaction

In this paper, three kinds of three-input logic gates are designed based on DNA strand displacement reaction, which are three-input OR logic gate, three-input AND logic gate, and three-input MAJORITY logic gate. The logic gates designed in this paper takes different DNA strands as input and fluorescence signals as output. The biochemical experimental results verify my designs. The results show that DNA strand displacement technology has important application value in DNA computing, especially in the construction of DNA molecular logic gates.

Parallel molecular computation on digital data stored in DNA

DNA is an incredibly dense storage medium for digital data. However, computing on the stored information is expensive and slow, requiring rounds of sequencing, in silico computation, and DNA synthesis. Prior work on accessing and modifying data using DNA hybridization or enzymatic reactions had limited computation capabilities. Inspired by the computational power of "DNA strand displacement," we augment DNA storage with "in-memory" molecular computation using strand displacement reactions to algorithmically modify data in a parallel manner. We show programs for binary counting and Turing universal cellular automaton Rule 110, the latter of which is, in principle, capable of implementing any computer algorithm. Information is stored in the nicks of DNA, and a secondary sequence-level encoding allows high-throughput sequencing-based readout. We conducted multiple rounds of computation on 4-bit data registers, as well as random access of data (selective access and erasure). We demonstrate that large strand displacement cascades with 244 distinct strand exchanges (sequential and in parallel) can use naturally occurring DNA sequence from M13 bacteriophage without stringent sequence design, which has the potential to improve the scale of computation and decrease cost. Our work merges DNA storage and DNA computing, setting the foundation of entirely molecular algorithms for parallel manipulation of digital information preserved in DNA.

Oxidative DNA damage on the VEGF G-quadruplex forming promoter is repaired via long-patch BER.

In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased ~two-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol β performs multiple insertions on G4 substrates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol β products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol β prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.

Small-Molecule Analysis Based on DNA Strand Displacement Using a Bacteriorhodopsin Photoelectric Transducer: Taking ATP as an Example.

A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.

Metal-mediated DNA strand displacement and molecular device operations based on base-pair switching of 5-hydroxyuracil nucleobases

Rational design of self-assembled DNA nanostructures has become one of the fastest-growing research areas in molecular science. Particular attention is focused on the development of dynamic DNA nanodevices whose configuration and function are regulated by specific chemical inputs. Herein, we demonstrate the concept of metal-mediated base-pair switching to induce inter- and intramolecular DNA strand displacement in a metal-responsive manner. The 5-hydroxyuracil (UOH) nucleobase is employed as a metal-responsive unit, forming both a hydrogen-bonded UOH–A base pair and a metal-mediated UOH–GdIII–UOH base pair. Metal-mediated strand displacement reactions are demonstrated under isothermal conditions based on the base-pair switching between UOH–A and UOH–GdIII–UOH. Furthermore, metal-responsive DNA tweezers and allosteric DNAzymes are developed as typical models for DNA nanodevices simply by incorporating UOH bases into the sequence. The metal-mediated base-pair switching will become a versatile strategy for constructing stimuli-responsive DNA nanostructures, expanding the scope of dynamic DNA nanotechnology.

Efficient Three-Dimensional DNA Nanomachine Guided by a Robust Tetrahedral DNA Nanoarray Structure for the Rapid and Ultrasensitive Electrochemical Detection of Matrix Metalloproteinase 2.

Herein, a giant-sized DNA nanoarray was subtly assembled by two kinds of independent tetrahedral DNA structures as the DNA track for a multi-armed three-dimensional (3D) DNA nanomachine to perform signal transduction and amplification efficiently, which was developed as an electrochemical biosensor for the rapid and ultrasensitive detection of matrix metalloproteinase 2 (MMP-2). Impressively, in contrast to conventional DNA walkers with inefficiency, which walked on random DNA tracks composed of a two-dimensional (2D) probe or a one-dimensional (1D) single-stranded (ss)DNA probe, the multi-armed 3D DNA nanomachine from exonuclease III (Exo III) enzyme-assisted target recycling amplification would be endowed with faster reaction speed and better walking efficiency because of the excellent rigidity and orderliness of the tetrahedral DNA nanoarray structure. Once the hairpin H3-label with the signal substance ferrocene (Fc) was added to the modified electrode surface, the multi-armed 3D DNA nanomachine would be driven to move along the well-designed nanoarray tracks by toehold-mediated DNA strand displacement, resulting in most of the ferrocene (Fc) binding to the electrode surface and a remarkable increase in electrochemical signals within 60 min. As a proof of concept, the prepared biosensor attained a low detection limit of 11.4 fg/mL for the sensitive detection of the target MMP-2 and was applied in Hela and MCF-7 cancer cell lysates. As a result, this strategy provided a high-performance sensing platform for protein detection in tumor diagnosis.

Programmable electrochemical biosensing platform based on catalytic hairpin assembly and entropy-driven catalytic cascade amplification circuit

Herein, powerful DNA strand displacement reaction and sensitive electrochemical analysis method were ingeniously integrated to develop a programmable biosensing platform. Using DNA as the detection model, a cascade amplification system based on catalytic hairpin assembly and entropy-driven catalytic was constructed, and the reaction rate and signal amplification effect were significantly improved. The product of the cascade amplification circuit could undergo strand displacement reaction with the signal probe on the electrode surface to obtain sensitive electrochemical signal changes and realize highly sensitive detection of the target. In addition, without redesigning the DNA sequences in the cascade amplification circuit, the by-product strand typically wasted in traditional entropy-driven catalytic reactions can be fully utilized to construct a single-signal output biosensing system and even a dual-signal output ratiometric biosensing platform, improving the detection repeatability and reliability of the system, and expanding the application of DNA strand displacement reaction in electrochemical biosensing. Furthermore, benefiting from the design flexibility of the DNA molecules, the constructed biosensing platform realized the sensitive detection of aptamer substrate (kanamycin as an example) and certain metal ion (mercury as an example) by simply recoding the corresponding recognition sequence, demonstrating the good versatility of the biosensing platform.

DNA-Based Signaling Networks for Transient Colloidal Co-Assemblies.

Programmable chemical circuits inspired by signaling networks in living cells are a promising approach for the development of adaptive and autonomous self-assembling molecular systems and material functions. Progress has been made at the molecular level, but connecting molecular control circuits to self-assembling larger elements such as colloids that enable real-space studies and access to functional materials is sparse and can suffer from kinetic traps, flocculation, or difficult system integration protocols. Herein, we report a toehold-mediated DNA strand displacement reaction network capable of autonomously directing two different microgels into transient and self-regulating co-assemblies. The microgels are functionalized with DNA and become elemental components of the network. The flexibility of the circuit design allows the installation of delay phases or accelerators by chaining additional circuit modules upstream or downstream of the core circuit. The design provides an adaptable and robust route to regulate other building blocks for advanced biomimetic functions.

Leveraging Steric Moieties for Kinetic Control of DNA Strand Displacement Reactions.

DNA strand displacement networks are a critical part of dynamic DNA nanotechnology and are proven primitives for implementing chemical reaction networks. Precise kinetic control of these networks is important for their use in a range of applications. Among the better understood and widely leveraged kinetic properties of these networks are toehold sequence, length, composition, and location. While steric hindrance has been recognized as an important factor in such systems, a clear understanding of its impact and role is lacking. Here, a systematic investigation of steric hindrance within a DNA toehold-mediated strand displacement network was performed through tracking kinetic reactions of reporter complexes with incremental concatenation of steric moieties near the toehold. Two subsets of steric moieties were tested with systematic variation of structures and reaction conditions to isolate sterics from electrostatics. Thermodynamic and coarse-grained computational modeling was performed to gain further insight into the impacts of steric hindrance. Steric factors yielded up to 3 orders of magnitude decrease in the reaction rate constant. This pronounced effect demonstrates that steric moieties can be a powerful tool for kinetic control in strand displacement networks while also being more broadly informative of DNA structural assembly in both DNA-based therapeutic and diagnostic applications that possess elements of steric hindrance through DNA functionalization with an assortment of chemistries.

Toeless and reversible DNA strand displacement based on Hoogsteen-bond triplex

Strand displacement reaction is a crucial component in the assembly of diverse DNA-based nanodevices, with the toehold-mediated strand displacement reaction representing the prevailing strategy. However, the single-stranded Watson-Crick sticky region that serves as the trigger for strand displacement can also cause leakage reactions by introducing crosstalk in complex DNA circuits. Here, we proposed the toeless and reversible DNA strand displacement reaction based on the Hoogsteen-bond triplex, which is compatible with most of the existing DNA circuits. We demonstrated that our proposed reaction can occur at pH 5 and can be reversed at pH 9. We also observed an approximately linear relationship between the degree of reaction and pH within the range of pH 5–6, providing the potential for precise regulation of the reaction. Meanwhile, by altering the sequence orientation, we have demonstrated that our proposed reaction can be initiated or regulated through the same toeless mechanism without the requirement for protonation in low pH conditions. Based on the proposed reaction principle, we further constructed a variety of DNA nanodevices, including two types of DNA logic gates that rely on pH 5/pH 9 changes for initiating and reversing: the AND gate and the OR gate. We also successfully constructed a DNA Walker based on our proposed reaction modes, which can move along a given track after the introduction of a programmable DNA sequence and complete a cycle after 4 steps. Our findings suggest that this innovative approach will have broad utility in the development of DNA circuits, molecular sensors, and other complex biological systems.

Enabling programmable dynamic DNA chemistry using small-molecule DNA binders

The binding of small molecules to the double helical structure of DNA, through either intercalation or minor groove binding, may significantly alter the stability and functionality of DNA, which is a fundamental basis for many therapeutic and sensing applications. Here, we report that small-molecule DNA binders can also be used to program reaction pathways of a dynamic DNA reaction, where DNA strand displacement can be tuned quantitatively according to the affinity, charge, and concentrations of a given DNA binder. The binder-induced nucleic acid strand displacement (BIND) thus enables innovative technologies to accelerate the discovery and characterization of bioactive small molecules. Specifically, we demonstrate the comprehensive characterization of existing and newly discovered DNA binders, where critical parameters for binding affinity and sequence selectivity can be obtained in a single, unbiased molecular platform without the need for any specialized equipment. We also engineer a tandem BIND system as a high-throughput screening assay for discovering DNA binders, through which 8 DNA binders were successfully discovered from a library of 700 compounds.

Washing-Free Electrochemiluminescence Biosensor for the Simultaneous Determination of N6 Methyladenosines Incorporating a Tri-Double Resolution Strategy.

We propose a novel washing-free electrochemiluminescence (ECL) biosensor for the simultaneous detection of two types of N6 methyladenosines-RNAs (m6A-RNAs), which are potential cancer biomarkers, on the basis of binding-induced DNA strand displacement (BINSD). The biosensor integrated a tri-double resolution strategy that combined spatial and potential resolution, hybridization and antibody recognition, and ECL luminescence and quenching. The biosensor was fabricated by separately immobilizing two ECL reagents (gold nanoparticles/g-C3N4 nanosheets and ruthenium bipyridine derivative/gold nanoparticles/Nafion) and the capture DNA probe on the two sections of glassy carbon electrode. As a proof of concept, m6A-Let-7a-5p and m6A-miR-17-5p were chosen as model analytes, while m6A antibody-DNA3/ferrocene-DNA4/ferrocene-DNA5 was designed as an m6A-binding probe and DNA6/DNA7 was designed as a hybridization probe with DNA3 to release the quenching probes ferrocene-DNA4/ferrocene-DNA5. The recognition process led to the quenching of the ECL signals from both probes via BINSD. The proposed biosensor has the advantage of being washing-free. The ECL methods using the fabricated ECL biosensor with the designed probes exhibited a low detection limit of 0.03 pM for two m6A-RNAs and high selectivity. This work reveals that this strategy is promising for developing an ECL method for the simultaneous detection of two m6A-RNAs. The proposed strategy could be expanded to develop the analytical methods for the simultaneous detection of other RNA modifications by changing the antibody and hybridization probe sequences.

Backstepping Synchronization Control for Three-dimensional Chaotic Oscillatory System via DNA Strand Displacement.

In previous work, a large number of articles have been published to implement coupled synchronization of chaotic systems in DNA-based reaction networks. Up to now, there have been few studies on backstepping synchronous control of chaotic systems through DNA strand displacement. A backstepping synchronization control approach for three-dimensional chaotic system by employing DNA strand displacement is developed in this study. First of all, using the programming properties of DNA molecules, four basic strand displacement reaction modules are given. In the light of these reaction modules as well as the law of mass action kinetics, a novel three-dimensional DNA chaotic system is presented. Second of all, by relying on backstepping control theory and DNA reaction modules, three synchronous controllers are developed to guarantee the synchronization between two three-dimensional DNA chaotic systems. Final of all, numerical simulation results are carried out to verify the validity and applicability of the backstepping synchronization control.

Front Cover: Enzyme‐Free Nucleic Acid Circuits for Fold‐Change Detection (ChemPlusChem 9/2023)

The cover picture shows an enzyme-free nucleic acid synthetic circuit constructed by the toehold-mediated DNA strand displacement reaction, where a fold-change input (top left) elicits a similar response (bottom right). This work is anticipated to shed new light on the design of DNA dynamic circuits. More information can be found in the Research Article by Hao Pei, Tong Zhu, and co-workers.