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

Abstract

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.

A Loser-Take-All DNA Circuit

“…Applying the estimated ks i and kr i in simulations, we predicted that the overall behavior of the loser-take-all circuit would bias toward identifying input X 2 as the smallest signal; this was indeed shown in experiments where output Z 2 turned ON the fastest when the concentration of X 2 was 0 (Figure a). It would be possible to reduce the difference in reaction rates by carefully redesigning the DNA sequences guided by sequence-level kinetics simulations, , but it would be challenging given the complexity of the circuit. We thus chose to explore the possibility of exploiting concentration adjustments to compensate for the rate differences.…”

A domain-level DNA strand displacement reaction enumerator allowing arbitrary non-pseudoknotted secondary structures

“…Future development of a more sophisticated rate model may adjust for the expected time spent in a reaction pathway [41], may incorporate nucleotide sequence, temperature and buffer conditions, and may be optimized via systematic parameter inference to better match experimental measurements. Eventually, such a kinetic model can complement thermodynamic energy parameters [73], and provide deeper insights into fundamental principles of nucleic acid folding.…”

“…For example, the stochastic nucleic acid sequence-level reaction simulator Multistrand [40] is suitable for estimating the rate of individual strand displacement reactions, but it cannot cope with the massive state space of a complex multistranded DSD system. However, Peppercorn can be used as a preprocessing step to enumerate a domain-level reaction network, and then the individual reaction rates can be calculated using sequence-level simulators [41].…”

“…We have implemented the Peppercorn enumerator in Python, available on GitHub [42], either as a standalone program for domain-level enumeration, or embeddable into other projects using the library interface. The peppercornenumerator library is already a central part of the DyNAMiC Workbench integrated development environment [43], the automated sequence-level verification software KinDA [41], and the 'CRN-to-DSD' compiler Nuskell [44]. Badelt et al [44] use Nuskell (and thus Peppercorn) to enumerate and compare 13 different DSD systems implementing a DNA-only oscillator [7].…”

Domain-Specific Programming Languages for Computational Nucleic Acid Systems

“…32 Thus, the Nuskell system provides one of the most integrated systems for the creation of DNA-based molecular programs that are correct by construction. The Peppercorn reaction enumerator has also been used in KinDA, 33 a tool for sequence-level analysis of the thermodynamics and kinetics of DNA strand displacement systems that can automatically detect deviations from the domain-level abstraction. This is an important direction for future research, as understanding the limits of the domain-level abstraction is a pressing concern for designers of nucleic acid circuits.…”