Automated Design of Programmable Enzyme-Driven DNA Circuits

Abstract

Molecular programming allows for the bottom-up engineering of biochemical reaction networks in a controlled in vitro setting. These engineered biochemical reaction networks yield important insight in the design principles of biological systems and can potentially enrich molecular diagnostic systems. The DNA polymerase-nickase-exonuclease (PEN) toolbox has recently been used to program oscillatory and bistable biochemical networks using a minimal number of components. Previous work has reported the automatic construction of in silico descriptions of biochemical networks derived from the PEN toolbox, paving the way for generating networks of arbitrary size and complexity in vitro. Here, we report an automated approach that further bridges the gap between an in silico description and in vitro realization. A biochemical network of arbitrary complexity can be globally screened for parameter values that display the desired function and combining this approach with robustness analysis further increases the chance of successful in vitro implementation. Moreover, we present an automated design procedure for generating optimal DNA sequences, exhibiting key characteristics deduced from the in silico analysis. Our in silico method has been tested on a previously reported network, the Oligator, and has also been applied to the design of a reaction network capable of displaying adaptation in one of its components. Finally, we experimentally characterize unproductive sequestration of the exonuclease to phosphorothioate protected ssDNA strands. The strong nonlinearities in the degradation of active components caused by this unintended cross-coupling are shown computationally to have a positive effect on adaptation quality.

High-resolution mapping of bifurcations in nonlinear biochemical circuits

“…This domination of M3 confirms predictions 40,42,43 that the sharing of limited resources, such as enzymes, introduces global and nonlinear couplings between substrates, which qualitatively impacts the dynamics of biochemical regulation networks, the 35 . The toolbox comprises three biochemical reactions: activation, inhibition (inh) and degradation.…”

Computer‐aided biochemical programming of synthetic microreactors as diagnostic devices

“…For more than decades, their unique information processing capabilities have fascinated both fundamental and engineering sciences (Feynman, ; Conrad, ; Bray, ). The field of synthetic biology has devoted considerable attention to expanding these biochemical mechanisms into scalable synthetic systems integrating modular biosensing and biocomputing with the hope to advance biotechnologies (Benenson et al , ; Benenson, ; Church et al , ; Pardee et al , ; Katz, ; Van Roekel et al , ; Pardee et al , ). Indeed, biomolecular machines capable of dynamic probing and decision‐making in situ could offer new ways to interface biology (Slomovic et al , ; Courbet et al , ) as well as unprecedented versatility in analytical and biomedical applications.…”

Boosting functionality of synthetic DNA circuits with tailored deactivation

“…We designed the sequences of the two autocatalytic subunits to avoid cross-binding, and the drain templates were constructed according to the rules introduced above. Relative concentrations were also inferred from the previous results, only keeping the total concentration of autocatalytic templates constant to mitigate enzyme load26. In addition, two fluorescent dyes were attached as N-quenching reporters25 on the activation templates in order to generate species-specific fluorescence reporting.…”

“…However, the precise kinetic laws of the chemical reactions connecting the network's nodes also play a fundamental role. In particular, the linear or nonlinear nature of these interactions are an essential determinant of the network's dynamics10262728.…”