RESUMO
Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.
Assuntos
Materiais Biocompatíveis , Hidrogéis , Animais , Hidrogéis/química , Biopolímeros , MamíferosRESUMO
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 substantial 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, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the 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.
Assuntos
Materiais Biocompatíveis , Biologia Sintética , PolímerosRESUMO
DNA-coated colloids can crystallize into a multitude of lattices, ranging from face-centered cubic to diamond, opening avenues to producing structures with useful photonic properties. The potential design space of DNA-coated colloids is large, but its exploration is hampered by a reliance on chemically modified DNA that is slow and expensive to commercially synthesize. Here we introduce a method to controllably tailor the sequences of DNA-coated particles by covalently appending new sequence domains onto the DNA grafted to colloidal particles. The tailored particles crystallize as readily and at the same temperature as those produced via direct chemical synthesis, making them suitable for self-assembly. Moreover, we show that particles coated with a single sequence can be converted into a variety of building blocks with differing specificities by appending different DNA sequences to them. This method will make it practical to identify optimal and complex particle sequence designs and paves the way to programming the assembly kinetics of DNA-coated colloids.
Assuntos
Coloides , DNA , DNA/química , Coloides/química , Temperatura , CinéticaRESUMO
The development of biomolecular stimuli-responsive hydrogels is important for biomimetic structures, soft robots, tissue engineering, and drug delivery. DNA polymerization gels are a new class of soft materials composed of polymer gel backbones with DNA duplex crosslinks that can be swollen by sequential strand displacement using hairpin-shaped DNA strands. The extensive swelling can be tuned using physical parameters such as salt concentration and biomolecule design. Previously, DNA polymerization gels have been used to create shape-changing gel automata with a large design space and high programmability. Here we systematically investigate how the swelling response of DNA polymerization gels can be tuned by adjusting the design and concentration of DNA crosslinks in the hydrogels or DNA hairpin triggers, and the ionic strength of the solution in which swelling takes place. We also explore the effect hydrogel size and shape have on the swelling response. Tuning these variables can alter the swelling rate and extent across a broad range and provide a quantitative connection between biochemical reactions and macroscopic material behaviour.
Assuntos
Hidrogéis , Cloreto de Sódio , Polimerização , Biomimética , DNARESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Trombina/química , Fator A de Crescimento do Endotélio Vascular/química , Técnicas Biossensoriais/métodos , Humanos , Ligação Proteica , Trombina/análise , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
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. Here we show how an ATP-dependent helicase, Rep-X, can drive specific dehybridization reactions at rates independent of sequence length, removing the constraints of equilibrium on DNA hybridization and dehybridization. To illustrate how this new capacity can speed up designed DNA reaction networks, 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.
Assuntos
DNA Catalítico , Pareamento de Bases , DNA/química , DNA Catalítico/metabolismo , Cinética , PolimerizaçãoRESUMO
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.
RESUMO
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.
Assuntos
Gelatina , Hidrogéis , Materiais Biocompatíveis , DNA , Humanos , PolimerizaçãoRESUMO
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.
Assuntos
Computadores Moleculares , DNA/química , Algoritmos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Engenharia Biomédica , Computadores Moleculares/estatística & dados numéricos , DNA/genética , DNA/ultraestrutura , Sistemas de Liberação de Medicamentos , Eletroquímica , Hidrogéis , Concentração de Íons de Hidrogênio , Nanocápsulas/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , NanotecnologiaRESUMO
The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/química , DNA/metabolismo , Nanotubos/química , Proteínas Virais/metabolismo , Sondas de DNA/química , RNA Polimerases Dirigidas por DNA/química , Regiões Promotoras Genéticas , RNA/metabolismo , Proteínas Virais/químicaRESUMO
Self-assembled DNA nanostructures have potential applications in therapeutics, diagnostics, and synthetic biology. A challenge in using DNA nanostructures in biological environments or cell culture, however, is that they may be degraded by enzymes found in these environments, such as nucleases. Such degradation can be slowed by introducing alternative substrates for nucleases, or by coating nanostructures with membranes or peptides. Here we demonstrate a means by which degradation can be reversed in situ through the repair of nanostructure defects. To demonstrate this effect, we show that degradation rates of DNA nanotubes, micron-scale self-assembled structures, are at least 4-fold lower in the presence of tiles that can repair nanotube defects during the degradation process. Micrographs of nanotubes show that tiles from solution incorporate into nanotubes and that this incorporation increases nanotube lifetime to several days in serum. We use experimental data to formulate a simple model of nanostructure self-healing. This model suggests how introducing even a relatively low rate of repair could allow a nanostructure to survive almost indefinitely because of a dynamic equilibrium between microscale repair and degradation processes. The ability to repair nanostructures could thus allow particular structures or devices to operate for long periods of time and might offer a single means to resist different types of chemical degradation.
Assuntos
DNA/química , Nanotubos/química , Polietilenoglicóis/química , Soro/metabolismo , Animais , Bovinos , DNA/metabolismo , Nanotecnologia , Nanotubos/ultraestrutura , Conformação de Ácido Nucleico , Polietilenoglicóis/metabolismoRESUMO
Self-assembly nanofabrication is increasingly appealing in complex nanostructures, as it requires fewer materials and has potential to reduce feature sizes. The use of DNA to control nanoscale and microscale features is promising but not fully developed. In this work, we study self-assembled DNA nanotubes to fabricate gold nanowires for use as interconnects in future nanoelectronic devices. We evaluate two approaches for seeding, gold and palladium, both using gold electroless plating to connect the seeds. These gold nanowires are characterized electrically utilizing electron beam induced deposition of tungsten and four-point probe techniques. Measured resistivity values for 15 successfully studied wires are between 9.3 × 10-6 and 1.2 × 10-3 Ωm. Our work yields new insights into reproducible formation and characterization of metal nanowires on DNA nanotubes, making them promising templates for future nanowires in complex electronic circuitry.
Assuntos
DNA/química , Nanopartículas Metálicas/química , Nanotubos/química , Nanofios/química , Ouro/química , Nanoestruturas/químicaRESUMO
A buffer reaction actively resists changes to the concentration of a chemical species. Typically, buffering reactions have only been able to regulate the concentration of hydronium (i.e., pH) and other ions. Here, we develop a new class of buffers that regulate the concentrations of short sequences of DNA (i.e., oligonucleotides). A buffer's behavior is determined by its set point concentration, capacity to resist disturbances, and response time after a disturbance. We provide simple mathematical formulas for selecting rate constants to tune each of these properties and show how to design DNA sequences and concentrations to implement the desired rate constants. We demonstrate several oligonucleotide buffers that maintain oligonucleotide set point concentrations between 10 and 80 nM in the presence of disturbances of 50 to 500 nM, with response times of less than 10 min to 1.5 h. Multiple buffers can regulate different sequences of DNA in parallel without crosstalk. Oligonucleotide buffers could stabilize and restore reactant concentrations in DNA circuits or in self-assembly processes, allowing such systems to operate reliably for extended durations. These buffers might also be coupled to other reactions to buffer molecules other than DNA. In general, an oligonucleotide buffer can be viewed as a chemical "battery" that maintains the total chemical potential of a buffered species in a closed system.
Assuntos
DNA/química , Oligodesoxirribonucleotídeos/química , Soluções Tampão , Hibridização de Ácido Nucleico , Fatores de TempoRESUMO
We investigate the kinetics and thermodynamics of DNA origami dimerization using flat rectangle origami components and different architectures of Watson-Crick complementary single-stranded DNA ("sticky end") linking strategies. We systematically vary the number of linkers, the length of the sticky ends on the linker, and linker architecture and measure the corresponding yields as well as forward and reverse reaction rate constants through fluorescence quenching assays. Yields were further verified using atomic force microscopy. We calculate values of H° and ΔS° for various interface designs and find nonlinear van't Hoff behavior, best described by two linear equations, suggesting distinct regimes of dimerization between those with and those without well-formed interfaces. We find that self-assembly reactions can be tuned by manipulating the interface architecture without suffering a loss in yield, even when yield is high, â¼75-80%. We show that the second-order forward reaction rate constant (k(on)) depends on both linker architecture and number of linkers used, with typical values on the order of 10(5)-10(6) (M·s)(-1), values that are similar to those of bimolecular association of small, complementary DNA strands. The k(on) values are generally non-Arrhenius, tending to increase with decreasing temperature. Finally, we use kinetic and thermodynamic information about the optimal linking architecture to extend the system to an infinite, two-component repeating lattice system and show that we can form micron-sized lattices, with well-formed structures up to 8 µm(2).
Assuntos
Pareamento de Bases , DNA/química , Conformação de Ácido Nucleico , Dimerização , Cinética , Microscopia de Força Atômica , Nanoestruturas/química , TermodinâmicaRESUMO
Understanding how a simple chemical system can accurately replicate combinatorial information, such as a sequence, is an important question for both the study of life in the universe and for the development of evolutionary molecular design techniques. During biological sequence replication, a nucleic acid polymer serves as a template for the enzyme-catalyzed assembly of a complementary sequence. Enzymes then separate the template and complement before the next round of replication. Attempts to understand how replication could occur more simply, such as without enzymes, have largely focused on developing minimal versions of this replication process. Here we describe how a different mechanism, crystal growth and scission, can accurately replicate chemical sequences without enzymes. Crystal growth propagates a sequence of bits while mechanically-induced scission creates new growth fronts. Together, these processes exponentially increase the number of crystal sequences. In the system we describe, sequences are arrangements of DNA tile monomers within ribbon-shaped crystals. 99.98% of bits are copied correctly and 78% of 4-bit sequences are correct after two generations; roughly 40 sequence copies are made per growth front per generation. In principle, this process is accurate enough for 1,000-fold replication of 4-bit sequences with 50% yield, replication of longer sequences, and darwinian evolution. We thus demonstrate that neither enzymes nor covalent bond formation are required for robust chemical sequence replication. The form of the replicated information is also compatible with the replication and evolution of a wide class of materials with precise nanoscale geometry such as plasmonic nanostructures or heterogeneous protein assemblies.
Assuntos
Cristalização , Replicação do DNA , Biocatálise , DNA/química , Evolução MolecularRESUMO
Control over when and where nanostructures arise is essential for the self-assembly of dynamic or multicomponent devices. We design and construct a DNA origami seed for the control of DAE-E tile DNA nanotube assembly. Seeds greatly accelerate nanotube nucleation and growth because they serve as nanotube nucleation templates. Seeds also control nanotube circumference. Simulations predict nanotube growth rates and suggest a small nucleation barrier remains when nanotubes grow from seeds.
Assuntos
DNA/química , Nanoestruturas/química , Nanotubos/química , DNA/síntese química , Microscopia de Força Atômica , Nanotecnologia , Conformação de Ácido NucleicoRESUMO
Synthetic biology is revolutionizing our approaches to biocomputing, diagnostics, and environmental monitoring through the use of designed genetic circuits that perform a function within a single cell. More complex functions can be performed by multiple cells that coordinate as they perform different subtasks. Cell-cell communication using molecular signals is particularly suited for aiding in this communication, but the number of molecules that can be used in different communication channels is limited. Here we investigate how proteases can limit the broadcast range of communicating cells. We find that adding barrierpepsin to Saccharomyces cerevisiae cells in two-dimensional multicellular networks that use α-factor signaling prevents cells beyond a specific radius from responding to α-factor signals. Such limiting of the broadcast range of cells could allow multiple cells to use the same signaling molecules to direct different communication processes and functions, provided that they are far enough from one another. These results suggest a means by which complex synthetic cellular networks using only a few signals for communication could be created by structuring a community of cells to create distinct broadcast environments.
Assuntos
Comunicação Celular , Saccharomyces cerevisiae , Transdução de Sinais , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Biologia Sintética/métodos , Peptídeo Hidrolases/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Bacteriophage RNA polymerases, in particular T7 RNA polymerase (RNAP), are well-characterized and popular enzymes for many RNA applications in biotechnology both in vitro and in cellular settings. These monomeric polymerases are relatively inexpensive and have high transcription rates and processivity to quickly produce large quantities of RNA. T7 RNAP also has high promoter-specificity on double-stranded DNA (dsDNA) such that it only initiates transcription downstream of its 17-base promoter site on dsDNA templates. However, there are many promoter-independent T7 RNAP transcription reactions involving transcription initiation in regions of single-stranded DNA (ssDNA) that have been reported and characterized. These promoter-independent transcription reactions are important to consider when using T7 RNAP transcriptional systems for DNA nanotechnology and DNA computing applications, in which ssDNA domains often stabilize, organize, and functionalize DNA nanostructures and facilitate strand displacement reactions. Here we review the existing literature on promoter-independent transcription by bacteriophage RNA polymerases with a specific focus on T7 RNAP, and provide examples of how promoter-independent reactions can disrupt the functionality of DNA strand displacement circuit components and alter the stability and functionality of DNA-based materials. We then highlight design strategies for DNA nanotechnology applications that can mitigate the effects of promoter-independent T7 RNAP transcription. The design strategies we present should have an immediate impact by increasing the rate of success of using T7 RNAP for applications in DNA nanotechnology and DNA computing.
Assuntos
RNA Polimerases Dirigidas por DNA , DNA , Nanoestruturas , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas Virais , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Nanoestruturas/química , DNA/metabolismo , DNA/genética , DNA/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/química , Nanotecnologia/métodos , Bacteriófago T7/genéticaRESUMO
Response interruption and redirection (RIRD) is a common treatment for automatically reinforced vocal stereotypy; it involves the contingent presentation of task instructions. Tasks that are included in RIRD are typically selected based on caregiver report, which may affect the efficacy of RIRD. The purpose of the current study was to evaluate the role of task preference in the efficacy of RIRD for four participants who engaged in vocal stereotypy. We conducted task-preference assessments and selected tasks of varying preferences to include in RIRD. For three out of four participants, the results showed that RIRD with higher preference tasks was not effective at reducing vocal stereotypy, whereas RIRD with lower preference tasks was effective for all participants.
Assuntos
Transtorno de Movimento Estereotipado , Voz , Humanos , Terapia Comportamental/métodos , Comportamento Estereotipado/fisiologia , Transtorno de Movimento Estereotipado/terapiaRESUMO
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, capable of enabling the construction of hydrogel architectures with tens to hundreds of different domains, presents a significant challenge. 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 µL. We also demonstrate that hydrogels fabricated using this platform exhibit responses similar to those of DNA-functionalized hydrogels fabricated using other methods by demonstrating that DNA sequences can hybridize within them and that they can undergo DNA sequence-induced shape change.