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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.

An important goal of synthetic biology is to build biosensors and circuits with well-defined input-output relationships that operate at speeds found in natural biological systems. However, for molecular computation, most commonly used genetic circuit elements typically involve several steps from input detection to output signal production: transcription, translation, and post-translational modifications. These multiple steps together require up to several hours to respond to a single stimulus, and this limits the overall speed and complexity of genetic circuits. To address this gap, molecular frameworks that rely exclusively on post-translational steps to realize reaction networks that can process inputs at a time scale of seconds to minutes have been proposed. Here, we build mathematical models of fast biosensors capable of producing Boolean logic functionality. We employ protease-based chemical and light-induced switches, investigate their operation, and provide selection guidelines for their use as on-off switches. As a proof of concept, we implement a rapamycin-induced switch

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.

Precise control over the nucleation, growth, and termination of self-assembly processes is a fundamental tool for controlling product yield and assembly dynamics. Mechanisms for altering these processes programmatically could allow the use of simple components to self-assemble complex final products or to design processes allowing for dynamic assembly or reconfiguration. Here we use DNA tile self-assembly to develop general design principles for building complexes that can bind to a growing biomolecular assembly and terminate its growth by systematically characterizing how different DNA origami nanostructures interact with the growing ends of DNA tile nanotubes. We find that nanostructures that present binding interfaces for all of the binding sites on a growing facet can bind selectively to growing ends and stop growth when these interfaces are presented on either a rigid or floppy scaffold. In contrast, nucleation of nanotubes requires the presentation of binding sites in an arrangement that matches the shape of the structure's facet. As a result, it is possible to build nanostructures that can terminate the growth of existing nanotubes but cannot nucleate a new structure. The resulting design principles for constructing structures that direct nucleation and termination of the growth of one-dimensional nanostructures can also serve as a starting point for programmatically directing two- and three-dimensional crystallization processes using nanostructure design.

An essential motif for the assembly of biological materials such as actin at the scale of hundreds of nanometers and beyond is a network of one-dimensional fibers with well-defined geometry. Here, we demonstrate the programmed organization of DNA filaments into micron-scale architectures where component filaments are oriented at preprogrammed angles. We assemble L-, T-, and Y-shaped DNA origami junctions that nucleate two or three micron length DNA nanotubes at high yields. The angles between the nanotubes mirror the angles between the templates on the junctions, demonstrating that nanoscale structures can control precisely how micron-scale architectures form. The ability to precisely program filament orientation could allow the assembly of complex filament architectures in two and three dimensions, including circuit structures, bundles, and extended materials.

The ability to detect a protein selectively and produce a predicted signal in real time is a long-lasting engineering challenge in the field of biochemistry. Such a mechanism typically requires a sensing module to recognize the input protein and a translation module to produce a programmable output signal that reflects the concentration of the input. Here we present a generic biomolecular reaction process that exchanges the concentration of an input protein with a DNA oligonucleotide. This approach uses the unique characteristic of DNA oligonucleotide aptamer that can either bind to a specific protein or to a complementary DNA oligonucleotide reversibly. We then pass the information of the protein concentration to the output signal through DNA strand displacement reactions. Using this strategy, we design and characterize four different exchange processes that can produce modular DNA oligonucleotides in response to different proteins such as clinically important human α-thrombin and vascular endothelial growth factor (VEGF). These exchange processes are capable of real time sensing and are modular such that they can be used for concurrent detection of different proteins with well-defined input-output characteristics. The novelty and simplicity of our approach encourage to develop advanced biochemical systems for point-of-care testing of infectious diseases and treatments.

An important goal of synthetic biology is to build biosensors and circuits with well-defined input-output relationships that operate at speeds found in natural biological systems. However, for molecular computation, most commonly used genetic circuit elements typically involve several steps from input detection to output signal production: transcription, translation, and post-translational modifications. These multiple steps together require up to several hours to respond to a single stimulus, and this limits the overall speed and complexity of genetic circuits. To address this gap, molecular frame-works that rely exclusively on post-translational steps to realize reaction networks that can process inputs at a timescale of seconds to minutes have been proposed. Here, we build mathematical models of fast biosensors capable of producing Boolean logic functionality. We employ protease-based chemical and light-induced switches, investigate their operation, and provide selection guidelines for their use as on-off switches. We then use these switches as elementary blocks, developing models for biosensors that can perform OR and XOR Boolean logic computation while using reaction conditions as tuning parameters. We use sensitivity analysis to determine the time-dependent sensitivity of the output to proteolytic and protein-protein binding reaction parameters. These fast protease-based biosensors can be used to implement complex molecular circuits with a capability of processing multiple inputs controllably and algorithmically. Our framework for evaluating and optimizing circuit performance can be applied to other molecular logic circuits.

Background Mucoid degeneration of the anterior cruciate ligament (ACL) is a common pathology but is often unknown and underdiagnosed. Mucinous material within the substance of ACL produces pain and limited motion in the knee. The purpose of this study was to diagnose mucoid degeneration of ACL and to assess the effectiveness of arthroscopic treatment in these patients. Materials and methods Between 2011 and 2014, 13 patients were diagnosed to be suffering from mucoid degeneration of ACL on the basis of magnetic resonance imaging (MRI), histopathology, and arthroscopy findings. All the patients had clinical symptoms of central knee pain behind patella and were unable to extend knees fully because of pain without instability. The aim of surgery was to remove as much of the degenerative mass as possible without having to sacrifice the entire ACL. Thus, the remaining ACL consisted of some intact anteromedial or posterolateral portion of the ACL interspersed with degenerate ACL tissue. Copious debridement of mucoid hypertrophied lesions of the ACL was performed. Results Mean follow-up was of 8.4 months (range 6–12 months) and all except one patient had a full range of painless motion. All patients have resumed their normal daily activities. None complained of any instability. Postoperatively, 12 knees showed complete pain relief and 1 showed pain improvement by at least 4 Visual Analogue Scale (VAS) grades and preoperative average International Knee Documentation Committee (IKDC) score 8 was 36.39 which improved postoperatively to the average 73.18. Conclusions Mucoid degeneration of the ACL should be suspected in patients presenting pain on terminal extension or flexion without preceding trauma. Prior knowledge of condition with high index of suspicion and careful interpretation of MRI can establish the diagnosis preoperatively. Arthroscopic debridement with or without notchplasty gives excellent functional results.

To build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.

Feedback allows biological systems to control gene expression precisely and reliably, even in the presence of uncertainty, by sensing and processing environmental changes. Taking inspiration from natural architectures, synthetic biologists have engineered feedback loops to tune the dynamics and improve the robustness and predictability of gene expression. However, experimental implementations of biomolecular control systems are still far from satisfying performance specifications typically achieved by electrical or mechanical control systems. To address this gap, we present mathematical models of biomolecular controllers that enable reference tracking, disturbance rejection, and tuning of the temporal response of gene expression. These controllers employ RNA transcriptional regulators to achieve closed loop control where feedback is introduced via molecular sequestration. Sensitivity analysis of the models allows us to identify which parameters influence the transient and steady state response of a target gene expression process, as well as which biologically plausible parameter values enable perfect reference tracking. We quantify performance using typical control theory metrics to characterize response properties and provide clear selection guidelines for practical applications. Our results indicate that RNA regulators are well-suited for building robust and precise feedback controllers for gene expression. Additionally, our approach illustrates several quantitative methods useful for assessing the performance of biomolecular feedback control systems.

A new analytical formulation for phase noise in MEMS oscillators was recently presented encompassing the role of essential nonlinearities in the electrical and mechanical domains. In this paper, we validate the effectiveness of the proposed analytical formulation with respect to the unified theory developed by Demir et al. describing phase noise in oscillators. In particular, it is shown that, over a range of the second-order mechanical nonlinear stiffness of the MEMS resonator, both models exhibit an excellent match in the phase diffusion coefficient calculation for a square-wave MEMS oscillator.

This paper presents the design, fabrication and characterization of a Variable Optical Attenuator (VOA). The VOA is based on a device known as the Mechanical Anti-reflection Switch (MARS) that has been well studied for modulation applications. Therefore, the requirements of the MEMS device need to be freshly ascertained and are done so in this paper taking the optical and MEMS parameters into account. In addition, the effect of the MEMS parameters on the optical ones and vise versa were also exploited in the design process. A good VOA should have a linear variation of attenuation with voltage and a large change in attenuation with a small change in voltage. In addition, the device should have as small an insertion loss as possible. Keeping this in mind, the dimensions of the device were chosen to achieve maximum performance. Four design variables were used to design MARS for the VOA application. This paper will discuss how they are related. A surface micro-machining fabrication method was developed using thermal evaporation and Plasma Enhanced Chemical Vapor Deposition (PECVD) techniques. It includes a fluoroform (CHF3) based reactive ion etching for silicon nitride. The devices were released using low surface tension liquids and Rapid Thermal Annealing (RTA). The achieved process yield was over 90%. Several devices were tested and attenuation up to 5.6 dB was observed experimentally. � 2011 Elsevier Ltd. All rights reserved.

We present a mathematical model of a microelectromechanical system (MEMS) oscillator that integrates the nonlinearities of the MEMS resonator and the oscillator circuitry in a single numerical modeling environment. This is achieved by transforming the conventional nonlinear mechanical model into the electrical domain while simultaneously considering the prominent nonlinearities of the resonator. The proposed nonlinear electrical model is validated by comparing the simulated amplitude-frequency response with measurements on an open-loop electrically addressed flexural silicon MEMS resonator driven to large motional amplitudes. Next, the essential nonlinearities in the oscillator circuit are investigated and a mathematical model of a MEMS oscillator is proposed that integrates the nonlinearities of the resonator. The concept is illustrated for MEMS transimpedance-amplifier- based square-wave and sine-wave oscillators. Closed-form expressions of steady-state output power and output frequency are derived for both oscillator models and compared with experimental and simulation results, with a good match in the predicted trends in all three cases.

Even though synchronization in autonomous systems has been observed for over three centuries, reports of systematic experimental studies on synchronized oscillators are limited. Here, we report on observations of internal synchronization in coupled silicon micromechanical oscillators associated with a reduction in the relative phase random walk that is modulated by the magnitude of the reactive coupling force between the oscillators. Additionally, for the first time, a significant improvement in the frequency stability of synchronized micromechanical oscillators is reported. The concept presented here is scalable and could be suitably engineered to establish the basis for a new class of highly precise miniaturized clocks and frequency references.

The modelling of the non-linear behaviour of MEMS oscillators is of interest to understand the effects of non-linearities on start-up, limit cycle behaviour and performance metrics such as output frequency and phase noise. This paper proposes an approach to integrate the non-linear modelling of the resonator, transducer and sustaining amplifier in a single numerical modelling environment so that their combined effects may be investigated simultaneously. The paper validates the proposed electrical model of the resonator through open-loop frequency response measurements on an electrically addressed flexural silicon MEMS resonator driven to large motional amplitudes. A square wave oscillator is constructed by embedding the same resonator as the primary frequency determining element. Measurements of output power and output frequency of the square wave oscillator as a function of resonator bias and driving voltage are consistent with model predictions ensuring that the model captures the essential non-linear behaviour of the resonator and the sustaining amplifier in a single mathematical equation.

In this paper, we demonstrate synchronization of two electrically coupled MEMS oscillators incorporating nearly identical silicon tuning fork microresonators. It is seen that as the output of the oscillators are coupled, they exhibit a synchronized response wherein the output amplitudes and signal-to-noise ratios of the two oscillators are improved relative to the case where the two oscillators are uncoupled. The observed output frequency of each oscillator before coupling is 219402.4 Hz and 219403.6 Hz respectively. In contrast, when the oscillators are driven simultaneously, they lock at a common output frequency of 219401.3 Hz and their outputs are found to be out-of-phase with respect to each other. A 6 dBm gain in output power and a reduction in the phase fluctuations of the output signal are observed for the coupled oscillators compared to the case when the oscillators are uncoupled.

The MEMS variable optical attenuator (VOA) presented in this work is an electrostatically controlled, surface micro-machined device. The device is designed, fabricated and optically characterized. The dimension of the device is designed to achieve maximum performance using COVENTORWARE software. A successful fabrication procedure is developed and used to realize the VOA. A releasing process is developed to release the device by using low surface tension liquids and rapid thermal annealing (RTA). The achieved process yield is over 90%. Several devices were tested and attenuation up to 5.6 dB was experimentally observed.

While many cellular processes are driven by biomolecular oscillators, precise control of a downstream on/off process by a biochemical oscillator signal can be difficult: over an oscillator's period, its output signal varies continuously between its amplitude limits and spends a significant fraction of the time at intermediate values between these limits. Further, the oscillator's output is often noisy, with particularly large variations in the amplitude. In electronic systems, an oscillating signal is generally processed by a downstream device such as a comparator that converts a potentially noisy oscillatory input into a square wave output that is predominantly in one of two well-defined on and off states. The comparator's output then controls downstream processes. We describe a method for constructing a synthetic biochemical device that likewise produces a square-wave-type biomolecular output for a variety of oscillatory inputs. The method relies on a separation of time scales between the slow rate of production of an oscillatory signal molecule and the fast rates of intermolecular binding and conformational changes. We show how to control the characteristics of the output by varying the concentrations of the species and the reaction rates. We then use this control to show how our approach could be applied to process different in vitro and in vivo biomolecular oscillators, including the p53-Mdm2 transcriptional oscillator and two types of in vitro transcriptional oscillators. These results demonstrate how modular biomolecular circuits could, in principle, be combined to build complex dynamical systems. The simplicity of our approach also suggests that natural molecular circuits may process some biomolecular oscillator outputs before they are applied downstream.

There has been much recent interest in engineering the phenomenon of synchronization in coupled micro-/nano-scale oscillators for applications ranging from precision time and frequency references to new approaches to information processing. This paper presents descriptive modelling detail and further experimental validation of the phenomenon of mutual synchronization in coupled MEMS oscillators building upon recent experimental validation of this concept by the present authors. In particular, the underlying dependence of the observation of synchronization on system parameters is studied through numerical and analytical modelling while considering essential nonlinearities in both the resonator and circuit domain. Experimental results demonstrating synchronized response are elaborated based on the realization of electrically coupled MEMS resonator based square-wave oscillators. The experimental results on frequency entrainment are found to be in general agreement with results obtained through analytical modeling and numerical simulation. The concept presented here is scalable and could be used to investigate the dynamics of large-arrays of coupled MEMS oscillators.