Standardized excitable elements for scalable engineering of far-from-equilibrium chemical networks

Abstract

Engineered far-from-equilibrium synthetic chemical networks that pulse or switch states in response to environmental signals could precisely regulate the kinetics of chemical synthesis or self-assembly pathways. Currently, such networks must be extensively tune to compensate for the different activities of and unintended reactions between a network's different chemical elements. Elements with standardized performance would allow rapid construction of networks with designed functions. Here we develop standardized excitable chemical elements, termed genelets, and use them to construct complex in vitro transcriptional networks. We develop a protocol for identifying >15 interchangeable genelet regulatory elements with uniform performance and minimal crosstalk. These elements can be combined to engineer feedforward and feedback modules whose dynamics are predicted by a simple kinetic model. We show modules can be rationally integrated and reorganized into networks that produce tunable temporal pulses and act as multi-state switchable memories. Standardized genelet elements, and the workflow to identify more, should make engineering complex far-from-equilibrium chemical dynamics routine.

Toehold-Mediated Strand Displacement in Random Sequence Pools

“…We refer to this as “internal toehold protection” (ITP) in the following. Similar approaches have already been used by other groups to protect strands from unwanted interactions. , Again, this technique limits the design space, as it necessitates potentially unwanted base pairing relationships within the circuit and may be incompatible with external sequence constraints. Ideally, one would like to use an approach that does not constrain the design space, while still making TMSD circuits robust with respect to an arbitrary sequence background.…”

Cascaded, Feedback-Driven, and Spatially Localized Emergence of Constitutional Dynamic Networks Driven by Enzyme-Free Catalytic DNA Circuits

“…The emergence of CDNs of diverse compositions and complexities demonstrated [2 × 2] CDN, [3 × 3] CDN, and 3D CDN that were synthesized by the enzyme-free CHA process and appropriately engineered hairpin motifs. Inspired by nature, where environmentally triggered complex dynamic networks in evolution of life and cellular transformations involve signal-triggered feedback processes and cascaded reactions, , we designed a two-layer cascaded emergence of CDNs employing a self-catalytic cascaded CHA circuit, as outlined in Figure A. In the parent system, eight hairpin motifs, H G , H G1 , H H , H H1 , H I , H I1 , H J , and H J1 , coexist as stable structures in the absence of primer P 3 .…”

“…These include adaptive 6,7 features, spatial confinement, 8 oscillatory, 9 feedback, 10 intercommunication between networks, 11 and the out-of-equilibrium dissipative operation of dynamic circuits. 12,13 Substantial research efforts are directed to the development of artificial biomimetic dynamic networks emulating functions of the native systems, 14 as a part of the rapidly advancing field of System Chemistry. 15−17 The information encoded in the base sequences of nucleic acids provides substantial structural and functional information into the biopolymer.…”

Toehold-Mediated Strand Displacement in Random Sequence Pools

“…Similar approaches have already been used by other groups to protect strands from unwanted interactions. 48,49 Again, this technique limits the design space, as it necessitates potentially unwanted base pairing relationships within the circuit and may be incompatible with external sequence constraints. Ideally, one would like to use an approach that does not constrain the design space, while still making TMSD circuits robust with respect to an arbitrary sequence background.…”