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The programmable spatiotemporal activation of molecules could be used to create stimuli-responsive biomaterials capable of executing multistage sensing and computational processes. Here we develop a method for the activation of different DNA molecules in precisely specified locations and times within hydrogels. The activation locations are determined by patterning molecules to be released in their inactive form within hydrogels at resolutions of tens of microns, whereas the time of activation is controlled by a DNA strand displacement reaction cascade that releases the target DNA oligonucleotides at precisely specified time intervals. This programmed activation of DNA from hydrogel substrates thus enables the scalable development of DNA-based reaction-diffusion systems that regulate DNA strand availability in space and time. This system could be used as a platform to autonomously activate predefined chemical signals embedded within a soft material.

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 tuned to compensate for the different activities of and unintended reactions between different network 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.

DNA-functionalized hydrogels are capable of sensing oligonucleotides, proteins, and small molecules, and specific DNA sequences sensed in the hydrogels environment can induce changes in these hydrogels shape and fluorescence. Fabricating DNA-functionalized hydrogel architectures with multiple domains could make it possible to sense multiple molecules and undergo more complicated macroscopic changes, such as changing fluorescence or changing the shapes of regions of the hydrogel architecture. However, automatically fabricating multi-domain DNA-functionalized hydrogel architectures, which could enable the construction of hydrogel architectures with tens to hundreds of different domains. We describe a platform for fabricating multi-domain DNA-functionalized hydrogels automatically at the micron scale, where reaction and diffusion processes can be coupled to program material behavior. Using this platform, the hydrogels material properties, such as shape and fluorescence, can be programmed, and the fabricated hydrogels can sense their environment. DNA-functionalized hydrogel architectures with domain sizes as small as 10 microns and with up to 4 different types of domains can be automatically fabricated using ink volumes as low as 50 microliters. We also demonstrate that hydrogels fabricated using this platform exhibit responses similar to those of DNA-functionalized gels fabricated using other methods by demonstrating that DNA sequences can hybridize within them and that they can undergo DNA sequence-induced shape change.

Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers, and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have significant implications to address grand challenges in health, biotechnology, and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have to date progressed mostly separately. This perspective article provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between these two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to “living” materials that sense and respond based on the reciprocal interactions between materials and embedded cells.

Controlling material shapes using information-bearing molecular signals is central to the creation of autonomous, reconfigurable soft devices. While physical and chemical stimuli can direct simple material swelling, bending, or folding, it has been challenging to direct multi-step shape-change programs crucial for complex, robotic tasks. Here, we demonstrate gel automata— sub-millimeter, photopatterned, highly swellable DNA gels—whose parts grow or shrink in response to easily designed DNA activator sequences, allowing for precisely controlled device articulation. We design and fabricate gel automata that reversibly transform between different letter shapes, and use neural networks to design automata that transform into every even or every odd numeral via designed reconfiguration programs. This sequential and repetitive metamorphosis of materials via chemical reorganization could dramatically advance our ability to manipulate micro-particles, cells, and tissues.One-Sentence SummaryPhotopatterned microgels follow sequences of DNA instructions to transform between complex, meaningful shapes such as letters and numerals.

Designed and engineered protein and DNA nanopores can be used to sense and characterize single molecules and control transmembrane transport of molecular species. However, designed biomolecular pores are less than 100 nm in length and are used primarily for transport across lipid membranes. Nanochannels that span longer distances could be used as conduits for molecules between nonadjacent compartments or cells. Here, we design micrometer-long, 7-nm-diameter DNA nanochannels that small molecules can traverse according to the laws of continuum diffusion. Binding DNA origami caps to channel ends eliminates transport and demonstrates that molecules diffuse from one channel end to the other rather than permeating through channel walls. These micrometer-length nanochannels can also grow, form interconnects, and interface with living cells. This work thus shows how to construct multifunctional, dynamic agents that control molecular transport, opening ways of studying intercellular signaling and modulating molecular transport between synthetic and living cells.

Molecular assemblies inside cells often undergo structural reconfiguration in response to stimuli to alter their function. Adaptive reconfiguration of cytoskeletal networks, for example, enables cellular shape change, movement, and cargo transport and plays a key role in driving complex processes such as division and differentiation. The cellular cytoskeleton is a self-assembling polymer network composed of simple filaments, so reconfiguration often occurs through the rearrangement of its component filaments' connectivities. DNA nanotubes have emerged as promising building blocks for constructing programmable synthetic analogs of cytoskeletal networks. Nucleating seeds can control when and where nanotubes grow, and capping structures can bind nanotube ends to stop growth. Such seeding and capping structures, collectively called termini, can organize nanotubes into larger architectures. However, these structures cannot be selectively activated or inactivated in response to specific stimuli to rearrange nanotube architectures, a key property of cytoskeletal networks. Here, we demonstrate how selective regulation of the binding affinity of DNA nanotube termini for DNA nanotube monomers or nanotube ends can direct the reconfiguration of nanotube architectures. Using DNA hybridization and strand displacement reactions that specifically activate or inactivate four orthogonal nanotube termini, we demonstrate that nanotube architectures can be reconfigured by selective addition or removal of distinct termini. Finally, we show how terminus activation could be a sensitive detector and amplifier of a DNA sequence signal. These results could enable the development of adaptive and multifunctional materials or diagnostic tools.

While many methods are available to measure the concentrations of proteins in solution, the development of a method to quantitatively report both increases and decreases in different protein concentrations in real-time using changes in the concentrations of other molecules, such as DNA outputs, has remained a challenge. Here, we present a biomolecular reaction process that reports the concentration of an input protein in situ as the concentration of an output DNA oligonucleotide strand. This method uses DNA oligonucleotide aptamers that bind either to a specific protein selectively or to a complementary DNA oligonucleotide reversibly using toehold-mediated DNA strand-displacement. It is possible to choose the sequence of output strand almost independent of the sensing protein. Using this strategy, we implemented four different exchange processes to report the concentrations of clinically relevant human α-thrombin and vascular endothelial growth factor using changes in concentrations of DNA oligonucleotide outputs. These exchange processes can operate in tandem such that the same or different output signals can indicate changes in concentration of distinct or identical input proteins. The simplicity of our approach suggests a pathway to build devices that can direct diverse output responses in response to changes in concentrations of specific proteins.

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.

Soft biomaterials possessing structural hierarchy have growing applications in lab-on-chip devices, artificial tissues, and micromechanical and chemomechanical systems. The ability to integrate sets of biomolecules, specifically DNA, within hydrogel substrates at precise locations could offer the potential to form and modulate complex biochemical processes with DNA-based molecular switches in such materials and provide a means of creating dynamic spatial patterns, thus enabling spatiotemporal control of a wide array of reaction-diffusion phenomena prevalent in biological systems. Here we develop a means of photopatterning two-dimensional DNA-functionalized poly(ethylene glycol) diacrylate (PEGDA) hydrogel architectures with an aim toward these applications. While PEGDA photopatterning methods are well-established for the fabrication of hydrogels, including those containing oligonucleotides, the photoinitiators typically used have significant crosstalk with many UV-photoswitchable chemistries including nitrobenzyl derivatives. We demonstrate the digital photopatterning of PEGDA

Techniques from structural DNA nanotechnology make it possible to assemble complex 3-dimensional nanostructures with virtually arbitrary control over their sizes, shapes and features at length scales of 3–100 nm, providing a flexible means for constructing nanoscale devices and machines. Here, we assemble micron-length DNA nanotubes and assess their performance as pipes for controlled ion transport. DNA nanotubes grow via assembly of DNA tiles from a seed pore, a 12-helix DNA origami cylinder functionalized with cholesterol, to form a DNA nanotube channel. The central channel of a nanotube can be obstructed via Watson-Crick hybridization of a channel cap, a second DNA origami structure, to the end of a nanotube channel or a nanotube seed pore. Single-channel electrophysiological characterization shows that both nanotube seed pores and nanotube channels display ohmic ion conductance consistent with their central channels’ diameters. Binding of the channel cap reduces the conductances of both DNA nanotube channels and seed pores, demonstrating control of ion-transport through these micron-length channels. Because these channels could be assembled into branched architectures or routed between specific molecular terminals, these results suggest a route to self-assembling nanofluidic devices and circuits in which transport can be controlled using dynamic biomolecular interactions.

In multicellular organisms, cells and tissues coordinate biochemical signal propagation across length scales spanning microns to meters. Endowing synthetic materials with similar capacities for coordinated signal propagation could allow these systems to adaptively regulate themselves across space and over time. Here we combine ideas from cell signaling and electronic circuitry to design a biochemical waveguide that transmits information in the form of a concentration of a DNA species on a directed path. The waveguide can be seamlessly integrated into a soft material because there is virtually no difference between the chemical or physical properties of the waveguide and the material it is embedded within. We propose the design of DNA strand displacement reactions to construct the system and, using reaction-diffusion models, identify kinetic and diffusive parameters that enable super-diffusive transport of DNA species via autocatalysis. Finally, to support experimental waveguide implementation, we show how a sink reaction could mitigate the spurious amplification of an autocatalyst within the waveguide, allowing for controlled waveguide triggering. Chemical waveguides could facilitate the design of synthetic biomaterials with distributed sensing machinery integrated throughout their structure and enable coordinated self-regulating programs triggered by changing environmental conditions.

The sequence-specific hybridization of DNA facilitates its use as a building block for designer nanoscale structures and reaction networks that perform computations. However, the strong binding energy of Watson-Crick base pairing that underlies this specificity also causes the DNA dehybridization rate to depend sensitively on sequence length and temperature. This strong dependency imposes stringent constraints on the design of multi-step DNA reactions, because a small deviation from the optimal conditions slows down the process dramatically. Here we show how an ATP-dependent helicase, Rep-X, can drive certain dehybridization reactions in designed DNA reaction networks at rates independent of sequence length, thereby decoupling the rates of hybridization and dehybridization. To illustrate this principle, we show that Rep-X extends the range of conditions where the primer exchange reaction, which catalytically adds a domain provided by a hairpin template to a DNA substrate, proceeds rapidly: in the strong substrate-hairpin binding regime, Rep-X expedites the reaction almost one hundred-fold. Our results provide an example of how ATP consumption can drive specific dehybridization reactions in designed DNA reaction networks and how this consumption can be harnessed to expedite reactions beyond their equilibrium rates.

DNA nanotechnology offers a route towards the synthesis of custom nano-structured materials and circuits through hierarchical assembly processes. While predictive kinetic models are being developed for the assembly of DNA nanostructures from small monomeric components, a general model for the hierarchical assembly of DNA nanostructures remains elusive. DNA tile nanotubes provide an ideal model system for the study of hierarchical assembly via end-to-end joining. In this study, we experimentally characterize the length-dependence of the end-to-end joining rate of DNA tile nanotubes. We then test the ability of three different models of polymer end-to-end joining to reproduce experimentally measured changes in nanotube lengths during a joining reaction using an ODE model for nanotube joining. All three models predict physically realistic joining rates that are consistent with prior measurements, with a length-independent end-to-end joining rate model providing the best fit to the experimental data. A length-independent constant joining rate is consistent with other DNA self-assembly processes across a broad range of length scales and also suggests how tractable models for hierarchical DNA nanostructure could be developed.

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.

Mesoscale molecular assemblies on the cell surface, such as cilia and filopodia, integrate information, control transport and amplify signals. Synthetic devices mimicking these structures could sensitively monitor these cellular functions and direct new ones. The challenges in creating such devices, however are that they must be integrated with cells in a precise kinetically controlled process and a device's structure and its precisely structured cell interface must then be maintained during active cellular function. Here we report the ability to integrate synthetic micro-scale filaments, DNA nanotubes, into a cell's architecture by anchoring them by their ends to specific receptors on the surfaces of mammalian cells. These filaments can act as shear stress meters: how anchored nanotubes bend at the cell surface quantitatively indicates the magnitude of shear stresses between 0-2 dyn per cm2, a regime important for cell signaling. Nanotubes can also grow while anchored to cells, thus acting as dynamic components of cells. This approach to cell surface engineering, in which synthetic biomolecular assemblies are organized within existing cellular architecture, could make it possible to build new types of sensors, machines and scaffolds that can interface with, control and measure properties of cells., 20 pages, 5 figures

Molecular assemblies inside cells often undergo structural reconfiguration in response to stimuli to alter their function. Adaptive reconfiguration of cytoskeletal networks, for example, enables cellular shape change, movement, and cargo transport and plays a key role in driving complex processes such as division and differentiation. The cellular cytoskeleton is a self-assembling polymer network composed of simple filaments, so reconfiguration often occurs through the rearrangement of its component filaments' connectivities. DNA nanotubes have emerged as promising building blocks for constructing programmable synthetic analogs of cytoskeletal networks. Nucleating seeds can control when and where nanotubes grow, and capping structures can bind nanotube ends to stop growth. Such seeding and capping structures, collectively called termini, can organize nanotubes into larger architectures. However, these structures cannot be selectively activated or inactivated in response to specific stimuli to rearrange nanotube architectures, a key property of cytoskeletal networks. Here, we demonstrate how selective regulation of the binding affinity of DNA nanotube termini for DNA nanotube monomers or nanotube ends can direct the reconfiguration of nanotube architectures. Using DNA hybridization and strand displacement reactions that specifically activate or inactivate four orthogonal nanotube termini, we demonstrate that nanotube architectures can be reconfigured by selective addition or removal of distinct termini. Finally, we show how terminus activation could be a sensitive detector and amplifier of a DNA sequence signal. These results could enable the development of adaptive and multifunctional materials or diagnostic tools.

Living systems can form and recover complex chemical patterns with precisely sized features in the ranges of tens or hundreds of microns. We show how designed reaction-diffusion processes can likewise produce precise patterns, termed attractor patterns, that reform their precise shape after being perturbed. We use oligonucleotide reaction networks, photolithography, and microfluidic delivery to form precisely controlled attractor patterns and study the responses of these patterns to different localized perturbations. Linear and "hill"-shaped patterns formed and stabilized into shapes and at time scales consistent with reaction-diffusion models. When patterns were perturbed in particular locations with UV light, they reliably reformed their steady-state profiles. Recovery also occurred after repeated perturbations. By designing the far-from-equilibrium dynamics of a chemical system, this study shows how it is possible to design spatial patterns of molecules that are sustained and regenerated by continually evolving towards a specific steady state configuration.

Hydrogels with the ability to change shape in response to biochemical stimuli are important for biosensing, smart medicine, drug delivery, and soft robotics. Here, a family of multicomponent DNA polymerization motor gels with different polymer backbones is created, including acrylamide-co-bis-acrylamide (Am-BIS), poly(ethylene glycol) diacrylate (PEGDA), and gelatin-methacryloyl (GelMA) that swell extensively in response to specific DNA sequences. A common mechanism, a polymerization motor that induces swelling is driven by a cascade of DNA hairpin insertions into hydrogel crosslinks. These multicomponent hydrogels can be photopatterned into distinct shapes, have a broad range of mechanical properties, including tunable shear moduli between 297 and 3888 Pa and enhanced biocompatibility. Human cells adhere to the GelMA-DNA gels and remain viable during ≈70% volumetric swelling of the gel scaffold induced by DNA sequences. The results demonstrate the generality of sequential DNA hairpin insertion as a mechanism for inducing shape change in multicomponent hydrogels, suggesting widespread applicability of polymerization motor gels in biomaterials science and engineering.