Predicting DNA kinetics with a truncated continuous-time Markov chain method.

Predicting the kinetics of reactions involving nucleic acid strands is a fundamental task in biology and biotechnology. Reaction kinetics can be modeled as an elementary step continuous-time Markov chain, where states correspond to secondary structures and transitions correspond to base pair formation and breakage. Since the number of states in the Markov chain could be large, rates are determined by estimating the mean first passage time from sampled trajectories. As a result, the cost of kinetic predictions becomes prohibitively expensive for rare events with extremely long trajectories. Also problematic are scenarios where multiple predictions are needed for the same reaction, e.g., under different environmental conditions, or when calibrating model parameters, because a new set of trajectories is needed multiple times. We propose a new method, called pathway elaboration, to handle these scenarios. Pathway elaboration builds a truncated continuous-time Markov chain through both biased and unbiased sampling. The resulting Markov chain has moderate state space size, so matrix methods can efficiently compute reaction rates, even for rare events. Also the transition rates of the truncated Markov chain can easily be adapted when model or environmental parameters are perturbed, making model calibration feasible. We illustrate the utility of pathway elaboration on toehold-mediated strand displacement reactions, show that it well-approximates trajectory-based predictions of unbiased elementary step models on a wide range of reaction types for which such predictions are feasible, and demonstrate that it performs better than alternative truncation-based approaches that are applicable for mean first passage time estimation. Finally, in a small study, we use pathway elaboration to optimize the Metropolis kinetic model of Multistrand, an elementary step simulator, showing that the optimized parameters greatly improve reaction rate predictions. Our framework and dataset are available at https://github.com/DNA-and-Natural-Algorithms-Group/PathwayElaboration.

Automated sequence-level analysis of kinetics and thermodynamics for domain-level DNA strand-displacement systems.

As an engineering material, DNA is well suited for the construction of biochemical circuits and systems, because it is simple enough that its interactions can be rationally designed using Watson-Crick base pairing rules, yet the design space is remarkably rich. When designing DNA systems, this simplicity permits using functional sections of each strand, called domains, without considering particular nucleotide sequences. However, the actual sequences used may have interactions not predicted at the domain-level abstraction, and new rigorous analysis techniques are needed to determine the extent to which the chosen sequences conform to the system's domain-level description. We have developed a computational method for verifying sequence-level systems by identifying discrepancies between the domain-level and sequence-level behaviour. This method takes a DNA system, as specified using the domain-level tool Peppercorn, and analyses data from the stochastic sequence-level simulator Multistrand and sequence-level thermodynamic analysis tool NUPACK to estimate important aspects of the system, such as reaction rate constants and secondary structure formation. These techniques, implemented as the Python package KinDA, will allow researchers to predict the kinetic and thermodynamic behaviour of domain-level systems after sequence assignment, as well as to detect violations of the intended behaviour.

Modelling DNA origami self-assembly at the domain level.

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

Guiding the folding pathway of DNA origami.

DNA origami is a robust assembly technique that folds a single-stranded DNA template into a target structure by annealing it with hundreds of short 'staple' strands. Its guiding design principle is that the target structure is the single most stable configuration. The folding transition is cooperative and, as in the case of proteins, is governed by information encoded in the polymer sequence. A typical origami folds primarily into the desired shape, but misfolded structures can kinetically trap the system and reduce the yield. Although adjusting assembly conditions or following empirical design rules can improve yield, well-folded origami often need to be separated from misfolded structures. The problem could in principle be avoided if assembly pathway and kinetics were fully understood and then rationally optimized. To this end, here we present a DNA origami system with the unusual property of being able to form a small set of distinguishable and well-folded shapes that represent discrete and approximately degenerate energy minima in a vast folding landscape, thus allowing us to probe the assembly process. The obtained high yield of well-folded origami structures confirms the existence of efficient folding pathways, while the shape distribution provides information about individual trajectories through the folding landscape. We find that, similarly to protein folding, the assembly of DNA origami is highly cooperative; that reversible bond formation is important in recovering from transient misfoldings; and that the early formation of long-range connections can very effectively enforce particular folds. We use these insights to inform the design of the system so as to steer assembly towards desired structures. Expanding the rational design process to include the assembly pathway should thus enable more reproducible synthesis, particularly when targeting more complex structures. We anticipate that this expansion will be essential if DNA origami is to continue its rapid development and become a reliable manufacturing technology.