On the biophysics and kinetics of toehold-mediated DNA strand displacement

Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical controls electromechanical devices, living organisms employ wiring to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry. In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive. Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the device physics of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for hybridization, fraying, and branch migration, and provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems. In Chapters 3 and 4, we identify and overcome the crucial experimental challenges involved in using our general DNA-based technology for engineering dynamical behaviors in the test tube. In this process, we identify important design rules that inform our choice of molecular motifs and our algorithms for designing and verifying DNA sequences for our molecular implementation. We also develop flexible molecular strategies for tuning our reaction rates and stoichiometries in order to compensate for unavoidable non-idealities in the molecular implementation, such as imperfectly synthesized molecules and spurious leak pathways that compete with desired pathways. We successfully implement three distinct autocatalytic reactions, which we then combine into a de novo chemical oscillator. Unlike biological networks, which use sophisticated evolved molecules (like proteins) to realize such behavior, our test tube realization is the first to demonstrate that Watson-Crick base pairing interactions alone suffice for oscillatory dynamics. Since our design pipeline is general and applicable to any CRN, our experimental demonstration of a de novo chemical oscillator could enable the systematic construction of CRNs with other dynamic behaviors.

Cooperative strand displacement circuit with dual-toehold and bulge-loop structure for single-nucleotide variations discrimination

In this thesis I present a novel coarse-grained model of deoxyribonucleic acid (DNA). The model represents single-stranded DNA as a chain of rigid nucleotides, and includes potentials to represent chain connectivity, excluded volume, hydrogen-bonding and base stacking interactions. The parameterization of these interactions is justified by comparing the model's representation of a range of physical phenomena to experimental data. In particular, the geometrical structure and elastic moduli of duplex DNA, and the flexibility of single-stranded DNA, are shown to be physically reasonable. Additionally, the thermodynamics of single-stranded stacking, duplex hybridization, hairpin formation and more complex motifs are shown to agree well with experimental data. The model is optimized for capturing the thermodynamic and mechanical changes associated with duplex formation from single strands. Considerable attention is therefore given to ensuring that single-stranded DNA behaves physically, an approach which differs from previous attempts to model DNA. As a result, the model is the first in which an explicit stacking transition is present in single strands, and also the only coarse-grained model to date to capture both hairpin formation within a single strand and duplex formation between strands. The scope of the model is demonstrated by simulating DNA tweezers, an iconic nanodevice -- the first time that coarse-grained modelling has been applied to dynamic DNA nanotechnology. The simulations suggest that branch migration during toehold-mediated strand displacement -- a central feature of many nanomachines -- does not have a flat free-energy profile, as is generally assumed. This finding may help to explain the observed dependence of displacement rate on toehold length. Finally, the operation of a two-footed DNA walker on a single-stranded DNA track is considered. The model suggests that several aspects of the walker will reduce its efficiency, including a tendency to bind to an undesired site on the track. Several design modifications are suggested to improve the operation of the walker.

Toehold-mediated strand displacement (TMSD) is extensively utilized in dynamic DNA nanotechnology and for a wide range of DNA or RNA-based reaction circuits. Investigation of TMSD kinetics typically relies on bulk fluorescence measurements providing effective, bulk-averaged reaction rates. Information on individual molecules or even base pairs is scarce. In this work, we explore the dynamics of strand displacement processes at the single-molecule level using single-molecule force spectroscopy with a microfluidics-enhanced optical trap supported by state-of-the-art coarse-grained simulations. By applying force, we can trigger and observe TMSD in real-time with microsecond and nanometer resolution. We find TMSD proceeds very rapidly under load with single step times of 1 µs. Tuning invasion efficiency by introducing mismatches allows studying thousands of forward/backward invasion events on a single molecule and analyze the kinetics of the invasion process. Extrapolation to zero force reveals single step times for DNA invading DNA four times faster than for RNA invading RNA. We also study the kinetics of DNA invading RNA, a process that in the absence of force would rarely occur. Our results reveal the importance of sequence effects for the TMSD process and have relevance for a wide range of applications in nucleic acid nanotechnology and synthetic biology.

Proteins belonging to the highly conserved RecQ helicase family are essential for the maintenance of genomic stability. Here, we describe the biochemical properties of the human RECQ5beta protein. Like BLM and WRN, RECQ5beta is an ATP-dependent 3'-5' DNA helicase that can promote migration of Holliday junctions. However, RECQ5beta required the single-stranded DNA-binding protein RPA in order to mediate the efficient unwinding of oligonucleotide-based substrates. Surprisingly, we found that RECQ5beta possesses an intrinsic DNA strand-annealing activity that is inhibited by RPA. Analysis of deletion variants of RECQ5beta revealed that the DNA helicase activity resides in the conserved N-terminal portion of the protein, whereas strand annealing is mediated by the unique C-terminal domain. Moreover, the strand-annealing activity of RECQ5beta was strongly inhibited by ATPgammaS, a poorly hydrolyzable analog of ATP. This effect was alleviated by mutations in the ATP-binding motif of RECQ5beta, indicating that the ATP-bound form of the protein cannot promote strand annealing. This is the first demonstration of a DNA helicase with an intrinsic DNA strand-annealing function residing in a separate domain.

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.

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.

Dynamic DNA nanotechnology, a subfield of DNA nanotechnology, is concerned with the study and application of nucleic acid strand-displacement reactions. Strand-displacement reactions generally proceed by three-way or four-way branch migration and initially were investigated for their relevance to genetic recombination. Through the use of toeholds, which are single-stranded segments of DNA to which an invader strand can bind to initiate branch migration, the rate with which strand displacement reactions proceed can be varied by more than 6 orders of magnitude. In addition, the use of toeholds enables the construction of enzyme-free DNA reaction networks exhibiting complex dynamical behavior. A demonstration of this was provided in the year 2000, in which strand displacement reactions were employed to drive a DNA-based nanomachine (Yurke, B.; et al. Nature 2000, 406, 605-608). Since then, toehold-mediated strand displacement reactions have been used with ever increasing sophistication and the field of dynamic DNA nanotechnology has grown exponentially. Besides molecular machines, the field has produced enzyme-free catalytic systems, all DNA chemical oscillators and the most complex molecular computers yet devised. Enzyme-free catalytic systems can function as chemical amplifiers and as such have received considerable attention for sensing and detection applications in chemistry and medical diagnostics. Strand-displacement reactions have been combined with other enzymatically driven processes and have also been employed within living cells (Groves, B.; et al. Nat. Nanotechnol. 2015, 11, 287-294). Strand-displacement principles have also been applied in synthetic biology to enable artificial gene regulation and computation in bacteria. Given the enormous progress of dynamic DNA nanotechnology over the past years, the field now seems poised for practical application.

Large branched DNA structures are constructed by two-step reassociation of separated complementary strands from restriction fragments of different lengths. The displacement of DNA strands initially annealed to longer complementary DNA sequences, a process mediated by branch migration, is very rapid and has thus far been followed only under conditions which are second order, DNA reassociation rate limiting. The average lifetime of branched DNA leading to displacement of 1.6 Kb strands is estimated to be less than 10 seconds under conditions of DNA reassociation, Tm-25 degrees C. Several DNA-binding drugs, including intercalating dyes, have been tested to determine their influence, if any, on the kinetics of DNA strand displacements by branch migration. Only actinomycin D was found to have significant effect under the conditions we have described. The kinetics of the strand displacement in the presence of low concentrations of actinomycin D remain second order and slower rate of strand displacement must be attributed to decreased rate of reassociation of DNA strands to form the branched intermediates. Consideration is given to the potential manipulation of DNA structures at site-directed branches and the limitations due to rapid strand displacements. The feasibility of constructing sufficiently large branched DNA regions to approach first order, branch migration rate limiting kinetics is also discussed.

Toehold-mediated strand displacement has proven extremely powerful in programming enzyme-free DNA circuits and DNA nanomachines. To achieve multistep, autonomous, and complex behaviors, toeholds must be initially inactivated by hybridizing to inhibitor strands or domains and then relieved from inactivation in a programmed, timed manner. Although powerful and reasonably robust, this strategy has several drawbacks that limit the architecture of DNA circuits. For example, the combination between toeholds and branch migration (BM) domains is 'hard wired' during DNA synthesis thus cannot be created or changed during the execution of DNA circuits. To solve this problem, I propose a strategy called 'associative toehold activation', where the toeholds and BM domains are connected via hybridization of auxiliary domains during the execution of DNA circuits. Bulged thymidines that stabilize DNA three-way junctions substantially accelerate strand displacement reactions in this scheme, allowing fast strand displacement initiated by reversible toehold binding. To demonstrate the versatility of the scheme, I show (1) run-time combination of toeholds and BM domains, (2) run-time recombination of toeholds and BM domains, which results in a novel operation 'toehold switching', and (3) design of a simple conformational self-replicator.

Measuring and Modeling the Effect of Single Mismatch on DNA Strand Displacement

The Effect of Basepair Mismatch on DNA Strand Displacement

Toehold-mediated strand displacement (TMSD) is a widely used process in dynamic DNA nanotechnology, which has been applied for the actuation of molecular devices, in biosensor applications, and for DNA-based molecular computation. Similar processes also occur in a biological context, when RNA strands invade secondary structures or duplexes of other RNA or DNA molecules. Complex reaction environments-inside cells or synthetic cells-potentially contain a large number of competing nucleic acid molecules that transiently bind to the components of the strand displacement reaction of interest and thus slow down its kinetics. Here, we investigate the kinetics of TMSD reactions compartmentalized into water-in-oil emulsion droplets-in both the presence and absence of a random sequence background-using a droplet microfluidic 'stopped flow' set-up. The set-up enables one to determine the kinetics within thousands of droplets and easily vary experimental parameters such as the stoichiometry of the TMSD components. While the average kinetics in the droplets coincides precisely with the bulk behaviour, we observe considerable variability among the droplets. This variability is partially explained by the encapsulation procedure itself, but appears to be more pronounced in reactions involving a random pool background.

DNA is used to construct synthetic systems that sense, actuate, move and compute. The operation of many dynamic DNA devices depends on toehold-mediated strand displacement, by which one DNA strand displaces another from a duplex. Kinetic control of strand displacement is particularly important in autonomous molecular machinery and molecular computation, in which non-equilibrium systems are controlled through rates of competing processes. Here, we introduce a new method based on the creation of mismatched base pairs as kinetic barriers to strand displacement. Reaction rate constants can be tuned across three orders of magnitude by altering the position of such a defect without significantly changing the stabilities of reactants or products. By modelling reaction free-energy landscapes, we explore the mechanistic basis of this control mechanism. We also demonstrate that oxDNA, a coarse-grained model of DNA, is capable of accurately predicting and explaining the impact of mismatches on displacement kinetics.

CRISPR (clustered regularly interspaced short palindromic repeats) technology has achieved great breakthroughs in terms of convenience and sensitivity; it is becoming the most promising molecular tool. However, only two CRISPR activation modes (single and double stranded) are available, and they have specificity and universality bottlenecks that limit the application of CRISPR technology in high-precision molecular recognition. Herein, we proposed a novel CRISPR/Cas12a unrestricted activation mode to greatly improve its performance. The new mode totally eliminates the need for a protospacer adjacent motif and accurately activates Cas12a through toehold-mediated strand displacement and branch migration, which is highly universal and ultra-specific. With this mode, we discriminated all mismatch types and detected the EGFR T790M and L858R mutations in very low abundance. Taken together, our activation mode is deeply incorporated with DNA nanotechnology and extensively broadens the application boundaries of CRISPR technology in biomedical and molecular reaction networks.