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

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. Currently, such networks must be extensively tuned to compensate for the different activities of and unintended reactions between a network's various chemical components. Modular elements with standardized performance could be used to rapidly construct networks with designed functions. Here we develop standardized excitable chemical regulatory elements, termed genelets, and use them to construct complex in vitro transcriptional networks. We develop a protocol for identifying >15 interchangeable genelet elements with uniform performance and minimal crosstalk. These elements can be combined to engineer feedforward and feedback modules whose dynamics match those predicted by a simple kinetic model. Modules can then be rationally integrated and organized into networks that produce tunable temporal pulses and act as multistate switchable memories. Standardized genelet elements, and the workflow to identify more, should make engineering complex far-from-equilibrium chemical dynamics routine.

Programming the Sequential Release of DNA.

This study presents a mechanism for releasing a series of different short DNA sequences from sequestered complexes, one after another, using coupled biochemical reactions. The process uses stages of coupled DNA strand-displacement reactions that first release an output molecule and then trigger the initiation of the next release stage. We demonstrate the sequential release of 25 nM of four different sequences of DNA over a day, both with and without a centralized "clock" mechanism to regulate release timing. We then demonstrate how the presence of a target input molecule can determine which of several different release pathways are activated, analogous to branching conditional statements in computer programming. This sequential release circuit offers a means to schedule downstream chemical events, such as steps in the assembly of a nanostructure, or stages in a material's response to a stimulus.