Peptides or peptide conjugates capable of assembling into one-dimensional (1D) nanostructures have been extensively investigated over the past two decades due to their implications in human diseases and also their interesting applications as biomaterials. While many of these filamentous assemblies contain a β-sheet-forming sequence as the key design element, their eventual morphology could assume a variety of shapes, such as fibrils, ribbons, belts, or cylinders. Deciphering the key factors that govern the stacking fashion of individual β-sheets will help understand the polymorphism of peptide assemblies and greatly benefit the development of functional materials from customized molecular design. Herein, we report the decisive role of electrostatic interactions in the lamination and untwisting of 1D assemblies of short peptides. We designed and synthesized three short peptides containing only six amino acids (EFFFFE, KFFFFK, and EFFFFK) to elucidate the effective control of β-sheet stacking. Our results clearly suggest that electrostatic repulsions between terminal charges reduce the pitch of the twisting β-sheet tapes, thus leading to highly twisted, intertwined fibrils or twisted ribbons, whereas reducing this repulsion, either through molecular design of peptide with opposite terminal charges or through coassembly of two peptides carrying opposite charges, results in formation of infinite assemblies such as belt-like morphologies. We believe these observations provide important insight into the generic design of β-sheet assemblies.

Soft materials that swell or change shape in response to external stimuli show extensive promise in regenerative medicine, targeted therapeutics, and soft robotics. Generally, a stimulus for shape change must interact directly with the material, limiting the types of stimuli that may be used and necessitating high stimulus concentrations. Here, we show how DNA strand-displacement controllers within hydrogels can mediate size change by interpreting, amplifying, and integrating stimuli and releasing signals that direct the response. These controllers tune the time scale and degree of DNA-crosslinked hydrogel swelling and can actuate dramatic material size change in response to <100 nM of a specific biomolecular input. Controllers can also direct swelling in response to small molecules or perform logic. The integration of these stimuli-responsive materials with biomolecular circuits is a major step towards autonomous soft robotic systems in which sensing and actuation are implemented by biomolecular reaction networks.

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.

The functional stability and lifetimes of synthetic molecular circuits in biological environments are important for long-term, stable sensors or controllers of cell or tissue behavior. DNA-based molecular circuits, in particular DNA strand-displacement circuits, provide simple and effective biocompatible control mechanisms and sensors, but are vulnerable to digestion by nucleases present in living tissues and serum-supplemented cell culture. The stability of double-stranded and single-stranded DNA circuit components in serum-supplemented cell medium and the corresponding effect of nuclease-mediated degradation on circuit performance were characterized to determine the major routes of degradation and DNA strand-displacement circuit failure. Simple circuit design choices, such as the use of 5' toeholds within the DNA complexes used as reactants in the strand-displacement reactions and the termination of single-stranded components with DNA hairpin domains at the 3' termini, significantly increase the functional lifetime of the circuit components in the presence of nucleases. Simulations of multireaction circuits, guided by the experimentally measured operation of single-reaction circuits, enable predictive realization of multilayer and competitive-reaction circuit behavior. Together, these results provide a basic route to increased DNA circuit stability in cell culture environments.

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.