Kinetics Of Strand Displacement Research Articles

Plug-and-Play Module for Reversible and Continuous Control of DNA Strand Displacement Kinetics.

Regulatory modules for controlling the kinetics of toehold-mediated strand displacement (TMSD) play critical roles in designing dynamic and dissipative DNA chemical reaction networks (CRNs) but are hardwired into sequence designs. Herein, we introduce antitoehold (At), a plug-and-play module for reversible and continuous tuning of TMSD kinetics by temporarily occupying the toehold domain via a metastable duplex and base stacking. We demonstrate that kinetic control can be readily activated or deactivated in real time for any TMSD by simply adding At or anti-At. Continuous tuning of TMSD kinetics can also be achieved by altering the concentration of At. Moreover, the simple addition of At could readily reprogram existing TMSDs into a pulse-generation DNA CRN with continuous tunability. Our At approach also offers a new way for engineering continuously tunable DNA hybridization probes, which may find practical uses for discriminating clinically important mutations. Because of the simplicity, we anticipate that At will find wide applications for engineering DNA CRNs with diverse dynamic and dissipative behaviors, and DNA hybridization probes with tunable affinity and selectivity.

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.

Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides.

Hybridization and strand displacement kinetics determine the evolution of the base paired configurations of mixtures of oligonucleotides over time. Although much attention has been focused on the thermodynamics of DNA and RNA base pairing in the scientific literature, much less work has been done on the time dependence of interactions involving multiple strands, especially in RNA. Here we provide a study of oligoribonucleotide interaction kinetics and show that it is possible to calculate the association, dissociation and strand displacement rates displayed by short oligonucleotides (5nt–12nt) that exhibit no expected secondary structure as simple functions of oligonucleotide length, CG content, ΔG of hybridization and ΔG of toehold binding. We then show that the resultant calculated kinetic parameters are consistent with the experimentally observed time dependent changes in concentrations of the different species present in mixtures of multiple competing RNA strands. We show that by changing the mixture composition, it is possible to create and tune kinetic traps that extend by orders of magnitude the typical sub-second hybridization timescale of two complementary oligonucleotides. We suggest that the slow equilibration of complex oligonucleotide mixtures may have facilitated the nonenzymatic replication of RNA during the origin of life.

Natural and Modified Oligonucleotide Sequences Show Distinct Strand Displacement Kinetics and These Are Affected Further by Molecular Crowders.

DNA and RNA strand exchange is a process of fundamental importance in biology. Herein, we used a FRET-based assay to investigate, for the first time, the stand exchange kinetics of natural DNA, natural RNA, and locked nucleic acid (LNA)-modified DNA sequences in vitro in PBS in the absence or presence of molecular additives and macromolecular crowders such as diethylene glycol dimethyl ether (deg), polyethylene glycol (peg), and polyvinylpyrrolidone (pvp). The results show that the kinetics of strand exchange mediated by DNA, RNA, and LNA-DNA oligonucleotide sequences are different. Different molecular crowders further affect the strand displacement kinetics, highlighting the complexity of the process of nucleic acid strand exchange as it occurs in vivo. In a peg-containing buffer, the rate constant of displacement was slightly increased for the DNA displacement strand, while it was slightly decreased for the RNA and the LNA-DNA strands compared with displacement in pure PBS. When we used a deg-containing buffer, the rate constants of displacement for all three sequences were drastically increased compared with displacement in PBS. Overall, we show that interactions of the additives with the duplex strands have a significant effect on the strand displacement kinetics and this effect can exceed the one exerted by the chemical nature of the displacement strand itself.

Natural and Modified Oligonucleotide Sequences Show Distinct Strand Displacement Kinetics and These Are Affected Further by Molecular Crowders

Natural and Modified Oligonucleotide Sequences Show Distinct Strand Displacement Kinetics and These Are Affected Further by Molecular Crowders

Kinetics of RNA and RNA:DNA Hybrid Strand Displacement.

In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.

Kinetics of heterochiral strand displacement from PNA-DNA heteroduplexes.

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.

First passage time study of DNA strand displacement

DNA strand displacement, in which a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule fluorescence resonance energy transfer approach termed the “fission” assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nucleotide displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence, except for T → U substitution. However, displacement times were largely insensitive to the monovalent salt concentration in the range of 0.25–1 M. Using a one-dimensional random walk model, we infer that the single-step displacement time is in the range of ∼30–300 μs, depending on the base identity. The framework presented here is broadly applicable to the kinetic analysis of multistep processes investigated at the single-molecule level.

Fine and bidirectional regulation of toehold-mediated DNA strand displacement by a wedge-like DNA tool.

A wedge-like tool was established to finely and bidirectionally regulate the displacement rate of toehold-mediated strand displacement. This novel tool is very robust in terms of sequence generality and is compatible with most of the current DNA nanodevices based on traditional toehold reactions.

Kinetics of Strand Displacement and Hybridization on Wireframe DNA Nanostructures: Dissecting the Roles of Size, Morphology, and Rigidity.

Dynamic wireframe DNA structures have gained significant attention in recent years, with research aimed toward using these architectures for sensing and encapsulation applications. For these assemblies to reach their full potential, however, knowledge of the rates of strand displacement and hybridization on these constructs is required. Herein, we report the use of single-molecule fluorescence methodologies to observe the reversible switching between double- and single-stranded forms of triangular wireframe DNA nanotubes. Specifically, by using fluorescently labeled DNA strands, we were able to monitor changes in intensity over time as we introduced different sequences. This allowed us to extract detailed kinetic information on the strand displacement and hybridization processes. Due to the polymeric nanotube structure, the ability to individually address each of the three sides, and the inherent polydispersity of our samples as a result of the step polymerization by which they are formed, a library of compounds could be studied independently yet simultaneously. Kinetic models relying on mono-exponential decays, multi-exponential decays, or sigmoidal behavior were adjusted to the different constructs to retrieve erasing and refilling kinetics. Correlations were made between the kinetic behavior observed, the site accessibility, the nanotube length, and the structural robustness of wireframe DNA nanostructures, including fully single-stranded analogs. Overall, our results reveal how the length, morphology, and rigidity of the DNA framework modulate the kinetics of strand displacement and hybridization as well as the overall addressability and structural stability of the structures under study.

Amplified detection of single base mismatches with the competing-strand assay reveals complex kinetic and thermodynamic behavior of strand displacement at the electrode surface

Detection of single-base mismatches with respect to a probe strand has been a predominant pursuit in electrochemical biosensor efforts, due to links found between single nucleotide polymorphisms (SNPs) and the predisposition to various diseases. We report an osmium tetroxide bipyridine-based, thermally-controlled, competitive-strand electrochemical assay to allow amplified detection of single-base mismatches. Optimally designed competitive-strand displacement and hybridization temperature allows us to distinguish the single-mismatched-target from the fully complementary sequence with unambiguous, highly reproducible, robust signal differences of over 90%. Furthermore, we find a complex interplay between the position of the redox label, variations in strand displacement kinetics due to mismatches incorporated into the competitive strand, and alterations in the melting temperature of DNA duplexes tethered on the gold surface, when probed by square-wave voltammetry. These insights will apply to any surface-tethered DNA-based electrochemical biosensor, and can help with understanding complex phenomena involved in these types of assays.

Accelerating DNA-Based Computing on a Supramolecular Polymer.

Dynamic DNA-based circuits represent versatile systems to perform complex computing operations at the molecular level. However, the majority of DNA circuits relies on freely diffusing reactants, which slows down their rate of operation substantially. Here we introduce the use of DNA-functionalized benzene-1,3,5-tricarboxamide (BTA) supramolecular polymers as dynamic scaffolds to template DNA-based molecular computing. By selectively recruiting DNA circuit components to a supramolecular BTA polymer functionalized with 10-nucleotide handle strands, the kinetics of strand displacement and strand exchange reactions were accelerated 100-fold. In addition, strand exchange reactions were also favored thermodynamically by bivalent interactions between the reaction product and the supramolecular polymer. The noncovalent assembly of the supramolecular polymers enabled straightforward optimization of the polymer composition to best suit various applications. The ability of supramolecular BTA polymers to increase the efficiency of DNA-based computing was demonstrated for three well-known and practically important DNA-computing operations: multi-input AND gates, Catalytic Hairpin Assembly and Hybridization Chain Reactions. This work thus establishes supramolecular BTA polymers as an efficient platform for DNA-based molecular operations, paving the way for the construction of autonomous bionanomolecular systems that confine and combine molecular sensing, computation, and actuation.

Exploration of the Kinetics of Toehold-Mediated Strand Displacement via Plasmon Rulers.

DNA/RNA strand displacement is one of the most fundamental reactions in DNA and RNA circuits and nanomachines. In this work, we reported an exploration of the dynamic process of the toehold-mediated strand displacement via core-satellite plasmon rulers at the single-molecule level. Applying plasmon rulers with unlimited lifetime, single-strand displacement triggered by the invader that resulted in stepwise leaving of satellite from the core was continuously monitored by changes of scattering signal for hours. The kinetics of strand displacement in vitro with three different toehold lengths have been investigated. Also, the study revealed the difference in the kinetics of strand displacement between DNA/RNA and DNA/DNA duplexes. For the kinetics study in vivo, influence from the surrounding medium has been evaluated using both phosphate buffer and cell lysate. Applying core-satellite plasmon rulers with high signal/noise ratio, kinetics study in living cells proceeded for the first time, which was not possible by conventional methods with a fluorescent reporter. The plasmon rulers, which are flexible, easily constructed, and robust, have proven to be effective tools in exploring the dynamical behaviors of biochemical reactions in vivo.

Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids.

Locked nucleic acids (LNAs) are conformationally restricted RNA nucleotides. Their increased thermal stability and selectivity toward their complements make them well-suited for diagnostic and therapeutic applications. Although the structural and thermodynamic properties of LNA-LNA, LNA-RNA, and LNA-DNA hybridizations are known, the kinetic effects of incorporating LNA nucleotides into DNA strand displacement systems are not. Here, we thoroughly studied the strand displacement kinetics as a function of the number and position of LNA nucleotides in DNA oligonucleotides. When compared to that of an all-DNA control, with an identical sequence, the leakage rate constant was reduced more than 50-fold, to an undetectable level, and the invasion rate was preserved for a hybrid DNA/LNA system. The total performance enhancement ratio also increased more than 70-fold when calculating the ratio of the invading rate to the leakage rate constants for a hybrid system. The rational substitution of LNA nucleotides for DNA nucleotides preserves sequence space while improving the signal-to-noise ratio of strand displacement systems. Hybrid DNA/LNA systems offer great potential for high-performance chemical reaction networks that include catalyzed hairpin assemblies, hairpin chain reactions, motors, walkers, and seesaw gates.

Single molecule based SNP detection using designed DNA carriers and solid-state nanopores.

Single nucleotide polymorphisms (SNPs) play a crucial role as molecular markers in medical and diagnostic application. We demonstrate a nanopore based method for SNP detection at the single molecule level. Designed DNA carriers are used to distinguish DNA strands containing only one single base difference and follow strand displacement kinetics.

Investigating the dynamics of surface-immobilized DNA nanomachines.

Surface-immobilization of molecules can have a profound influence on their structure, function and dynamics. Toehold-mediated strand displacement is often used in solution to drive synthetic nanomachines made from DNA, but the effects of surface-immobilization on the mechanism and kinetics of this reaction have not yet been fully elucidated. Here we show that the kinetics of strand displacement in surface-immobilized nanomachines are significantly different to those of the solution phase reaction, and we attribute this to the effects of intermolecular interactions within the DNA layer. We demonstrate that the dynamics of strand displacement can be manipulated by changing strand length, concentration and G/C content. By inserting mismatched bases it is also possible to tune the rates of the constituent displacement processes (toehold-binding and branch migration) independently, and information can be encoded in the time-dependence of the overall reaction. Our findings will facilitate the rational design of surface-immobilized dynamic DNA nanomachines, including computing devices and track-based motors.

Engineering a robust DNA split proximity circuit with minimized circuit leakage.

DNA circuit is a versatile and highly-programmable toolbox which can potentially be used for the autonomous sensing of dynamic events, such as biomolecular interactions. However, the experimental implementation of in silico circuit designs has been hindered by the problem of circuit leakage. Here, we systematically analyzed the sources and characteristics of various types of leakage in a split proximity circuit which was engineered to spatially probe for target sites held within close proximity. Direct evidence that 3′-truncated oligonucleotides were the major impurity contributing to circuit leakage was presented. More importantly, a unique strategy of translocating a single nucleotide between domains, termed ‘inter-domain bridging’, was introduced to eliminate toehold-independent leakages while enhancing the strand displacement kinetics across a three-way junction. We also analyzed the dynamics of intermediate complexes involved in the circuit computation in order to define the working range of domain lengths for the reporter toehold and association region respectively. The final circuit design was successfully implemented on a model streptavidin-biotin system and demonstrated to be robust against both circuit leakage and biological interferences. We anticipate that this simple signal transduction strategy can be used to probe for diverse biomolecular interactions when used in conjunction with specific target recognition moieties.

The Effect of Basepair Mismatch on DNA Strand Displacement

DNA strand displacement is a key reaction in DNA homologous recombination and DNA mismatch repair and is also heavily utilized in DNA-based computation and locomotion. Despite its ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Here, we measured toehold-mediated strand displacement kinetics using single-molecule fluorescence in the presence of a single basepair mismatch. The apparent displacement rate varied significantly when the mismatch was introduced in the invading DNA strand. The rate generally decreased as the mismatch in the invader was encountered earlier in displacement. Our data indicate that a single base pair mismatch in the invader stalls branch migration and displacement occurs via direct dissociation of the destabilized incumbent strand from the substrate strand. We combined both branch migration and direct dissociation into a model, which we term the concurrent displacement model, and used the first passage time approach to quantitatively explain the salient features of the observed relationship. We also introduce the concept of splitting probabilities to justify that the concurrent model can be simplified into a three-step sequential model in the presence of an invader mismatch. We expect our model to become a powerful tool to design DNA-based reaction schemes with broad functionality.

DNA and DNA computation based on toehold-mediated strand-displacement reactions

biocompatibility. Considering the critical role of DNA less than 150 base pairs (bp) in cellular processes such as regulated gene expression, quantifying the intrinsic bend ability of DNA on a sub-persistence length scale is essential to understanding its molecular functions and the DNA-protein interaction. From the classical point of view, double-stranded DNA is assumed to be stiff and can be treated by semi-flexible chain, but recent studies have yielded contradictory results. A lot of studies tried to prove that the worm-like chain model can be used to fully describe DNA chain. However, recent theoretical and experimental studies indicated that DNA exhibits high flexibility on a short length scale, which cannot be described by the worm-like chain model. Further studies are needed to address the extreme flexibility of DNA on a short length scale. On the basis of the predictability of the double helical structure and the Watson-Crick binding thermodynamics for DNA, a class of DNA reactions can be defined, called toehold-mediated strand-displacement reaction, in which one complementary single-stranded DNA sequence first binds to the dangling toehold domain of the substrate in a pre-hybridized double-stranded DNA, then triggers the strand-displacement reaction, and finally results in the dissociation of the third strand previously bound to the substrate with partial complementarity. In dynamic DNA nanotechnology, isothermal toehold-mediated DNA strand-displacement reaction has been used to design complex nanostructure and nanodevice for molecular computation. The kinetics of the strand-displacement can be modulated using the toehold length. In order to weaken the coupling between the kinetics of strand-displacement and the thermodynamics of the reaction, the concept of toehold exchange was introduced by Winfree et al. to improve the control of strand-displacement kinetics. More importantly, the biomolecular reaction (BM) rate constant of toehold exchange can be analytically derived using the three-step model. Through utilizing strand-displacement reactions and taking advantage of its programmable sequences and precise recognition properties, DNA can be used to build complex circuits which can proceed robustly at constant temperature, achieving specific functions. DNA strand-displacement reaction can be employed to fabricate logic gates, and large and complex circuits for DNA computing, to mimic the naturally occurring occurrence of biological systems. Based on that, DNA circuit can then be used to direct the assembly of nanodevice following the designed pathway, and modulate the chemical reaction networks on the surface of living cell or in cellular systems for biosensing, even program the cellular machinery in the future for genetic diagnostic or gene therapy. In the present paper, we reviewed the proceedings in the fields of DNA structure and conformational changes, and DNA flexibility, discussed the mechanism of DNA strand-displacement reaction at the molecular level, and introduced the recent studies in DNA computation as well as the dynamic DNA nanotechnology, such as self-assembly.

Modulating the DNA strand-displacement kinetics with the one-sided remote toehold design for differentiation of single-base mismatched DNA

A one-sided remote toehold design was proposed to provide the fine control over strand-displacement reaction kinetics with simplicity and versatility.