Chemical circuits can coordinate elaborate sequences of events in cells and tissues, from the self-assembly of biological complexes to the sequence of embryonic development. However, autonomously directing the timing of events in synthetic systems using chemical signals remains challenging. Here we demonstrate that a simple synthetic DNA strand-displacement circuit can release target sequences of DNA into solution at a constant rate after a tunable delay that can range from hours to days. The rates of DNA release can be tuned to the order of 1-100 nM per day. Multiple timer circuits can release different DNA strands at different rates and times in the same solution. This circuit can thus facilitate precise coordination of chemical events in vitro without external stimulation.

In recent years, a diverse set of mechanisms have been developed that allow DNA strands with specific sequences to sense information in their environment and to control material assembly, disassembly, and reconfiguration. These sequences could serve as the inputs and outputs for DNA computing circuits, enabling DNA circuits to act as chemical information processors to program complex behavior in chemical and material systems. This review describes processes that can be sensed and controlled within such a paradigm. Specifically, there are interfaces that can release strands of DNA in response to chemical signals, wavelengths of light, pH, or electrical signals, as well as DNA strands that can direct the self-assembly and dynamic reconfiguration of DNA nanostructures, regulate particle assemblies, control encapsulation, and manipulate materials including DNA crystals, hydrogels, and vesicles. These interfaces have the potential to enable chemical circuits to exert algorithmic control over responsive materials, which may ultimately lead to the development of materials that grow, heal, and interact dynamically with their environments.

Cells use sophisticated, multiscale spatial patterns of chemical instructions to control cell fate and tissue growth. While some types of synthetic pattern formation have been well studied1-6, it remains unclear how to design chemical processes that can reproducibly create similar spatial patterns. Here we describe a scalable approach for the design of processes that generate such patterns, which can be implemented using synthetic DNA reaction-diffusion networks7,8. In our method, black-box modules are connected together into integrated programs for arbitrarily complex pattern formation. These programs can respond to input stimuli, process information, and ultimately produce stable output patterns that differ in size and concentration from their inputs. To build these programs, we break a target pattern into a set of patterning subtasks, design modules to perform these subtasks independently, and combine the modules into networks. We demonstrate in simulation how programs designed with our methodology can generate complex patterns, including a French flag and a stick figure.

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.

We demonstrate reaction-diffusion systems that generate stable patterns of DNA oligonucleotide concentrations within agarose gels, including linear and "hill" (i.e. increasing then decreasing) shapes in one and two dimensions. The reaction networks that produce these patterns are driven by enzyme-free DNA strand-displacement reactions, in which reactant DNA complexes continuously release and recapture target strands of DNA in the gel; a balance of these reactions produces stable patterns. The reactant complexes are maintained at high concentrations by liquid reservoirs along the gel boundary. We monitor our patterns using time-lapse fluorescence microscopy and show that the shape of our patterns can be easily tuned by manipulating the boundary reservoirs. Finally, we show that two overlapping, stable gradients can be generated by designing two sets of non-interacting release and recapture reactions with DNA strand-displacement systems. This paper represents a step toward the generation of scalable, complex reaction-diffusion patterns for programming the spatiotemporal behavior of synthetic materials.