Cascade Circuit Research Articles

DNA Nanotechnology for Application in Targeted Protein Degradation.

DNA is a kind of flexible and versatile biomaterial for constructing nanostructures and nanodevices. Due to high biocompatibility and programmability and easy modification and fabrication, DNA nanotechnology has emerged as a powerful tool for application in intracellular targeted protein degradation. In this review, we summarize the recent advances in the design and mechanism of targeted protein degradation technologies such as protein hydrolysis targeted chimeras, lysosomal targeted chimeras, and autophagy based protein degradation. Subsequently, we introduce the DNA nanotechnologies of DNA cascade circuits, DNA nanostructures, and dynamic machines. Moreover, we present the latest developments in DNA nanotechnologies in targeted protein degradation. Finally, the vision and challenges are discussed.

On-Site Multiply Stimulated Self-Confinement of an Integrated DNA Cascade Circuit for Highly Reliable Intracellular Imaging of miRNA and In Situ Interrogation of the Relevant Regulatory Pathway.

Artificial DNA circuits represent a versatile yet promising toolbox for in situ monitoring and concomitant regulation of diverse biological events within live cells. Nonetheless, their performance is significantly impeded by the diffusion-dominated slow reaction kinetics and the uncontrollable off-target activation. Herein, a self-localized cascade (SLC) circuit is reported for the robust and efficient microRNA (miRNA) analysis in living cells. The SLC circuit consists of the cell-specific localization module and the analyte-specific signal amplification module. By integrating the reaction probes of these two modules, the complexity of the system is reduced to realize the responsive co-localization of circuitry probes and the simultaneous cascade signal amplification. Taking advantage of the specifically activated, self-localized, and cascade design, the SLC circuit successfully achieves the robust miRNA-21 (miR-21) imaging and the accurate cells differentiation. Moreover, the reverse regulation mechanism is successfully explored between messenger RNA (mRNA) and miRNA through the engineered SLC circuit and further elucidates the underlying signaling pathways between them. Therefore, the SLC circuit provides a powerful tool for the sensitive detection of intracellular biomolecules and the study of the corresponding cell regulatory mechanisms.

Potential pharmaceuticals targeting neuroimmune interactions in treating acute lung injury.

Although interactions between the nervous and immune systems have been recognized decades ago, it has become increasingly appreciated that neuroimmune crosstalk is among the driving factors of multiple pulmonary inflammatory diseases including acute lung injury (ALI). Here, we review the current understanding of nerve innervations towards the lung and summarize how the neural regulation of immunity and inflammation participates in the onset and progression of several lung diseases, especially ALI. We then present advancements in the development of potential drugs for ALI targeting neuroimmune interactions, including cholinergic anti-inflammatory pathway, sympathetic-immune pathway, purinergic signalling, neuropeptides and renin-angiotensin system at different stages from preclinical investigation to clinical trials, including the traditional Chinese medicine. This review highlights the importance of considering the therapeutic strategy of inflammatory diseases within a conceptual framework that integrates classical inflammatory cascade and neuroimmune circuits, so as to deepen the understanding of immune modulation and develop more sophisticated approaches to treat lung diseases represented by ALI. The lungs present abundant nerve innervations. Neuroimmune interactions exert a modulatory effect in the onset and progression of lung inflammatory diseases, especially acute lung injury. The advancements of potential drugs for ALI targeting neuroimmune crosstalk at different stages from preclinical investigation to clinical trials are elaborated. Point out the direction for the development of neuroimmune pharmacology in the future.

Stochastic response of ultrasensitivity: Optimized switching of mitogen activated protein kinase (MAPK) cascade

The intricate dynamics of ultrasensitive transitions within mitogen-activated protein kinase (MAPK) cascade circuits regulate the diverse physiological activities in a living organism emphasizing their pivotal role in cellular signal transduction. We have shown that the generic features of ultrasensitive transition often characterized through critical slowing down, however, one can have a theoretical estimate of such catastrophic transition using a stochastic response function, called early warning signals (EWS). EWS is composed of slowing down of decay of correlation and divergence of fluctuation phenomenon under very low perturbation. It is shown that the ultrasensitive transition arises depending on the trade off between the cycle number and essential activation parameter. Therefore, the transition point is kinetically optimized by these two parameters so that a high activation energy is surmounted through multiple steps and here we have shown that EWS as stochastic response functions can capture this optimization most effectively.

Movable toehold for leakless self-assembly circuits

Nonenzymatic self-assembly circuit utilizing hairpin substrates has been developed to be a powerful tool for information transduction, amplification and computation. However, the sensitivity, stability and application of this circuit are impeded by the presence of leakage which refers to undesired triggering in the absence of input. Herein, we proposed a movable toehold principle to suppress leakage and accelerate the catalytic reaction through removing partial hairpin toehold responsible for the leakage and transferring it to the catalyst. With movable toehold, catalytic hairpin assembly (called mtCHA) exhibited an excellent signal-to-background ratio of over 100, high robustness and improved specificity. In more complex circuit, including proximity recognition, signal amplification of small molecules (such as ATP), logic network, autocatalysis circuit and two-layer cascade circuit, mtCHA also demonstrated satisfactory performance. Our findings suggest that mtCHA holds great potential for broader applications, and the approach of repurposing harmful fragments into beneficial candidates can provide valuable insights for other chemical systems.

Construction of cascade circuits for dynamic temporal regulation and its application to PHB production

BackgroundTo maximize the production capacity and yield of microbial cell factories, metabolic pathways are generally modified with dynamic regulatory strategies, which can effectively solve the problems of low biological yield, growth retardation and metabolic imbalance. However, the strategy of dynamic regulating multiple genes in different time and order is still not effectively solved. Based on the quorum-sensing (QS) system and the principle of cascade regulation, we studied the sequence and time interval of gene expression in metabolic pathways.ResultsWe designed and constructed a self-induced dynamic temporal regulatory cascade circuit in Escherichia coli using the QS system and dual regulatory protein cascade and found that the time intervals of the cascade circuits based on the Tra, Las system and the Lux, Tra system reached 200 min and 150 min, respectively. Furthermore, a dynamic temporal regulatory cascade circuit library with time intervals ranging from 110 to 310 min was obtained based on this circuit using promoter engineering and ribosome binding site replacement, which can provide more selective synthetic biology universal components for metabolic applications. Finally, poly-β-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of the cascade circuit library. The content of PHB increased 1.5-fold. Moreover, circuits with different time intervals and different expression orders were found to have different potentials for application in PHB production, and the preferred time-interval circuit strain C2-max was identified by screening.ConclusionsThe self-induced dynamic temporal regulation cascade circuit library can enable the expression of target genes with sequential changes at different times, effectively solving the balance problem between cell growth and product synthesis in two-stage fermentation and expanding the application of dynamic regulatory strategies in the field of metabolic engineering.

Hairpin cascade circuits on programmable DNA-nucleated spherical nucleic acid for intracellular targeted microRNA imaging

DNA molecular probes have been increasingly used for biological imaging and tumor theranostics. In particular, live-cell imaging of low abundance microRNA (miRNA) with high accuracy and efficiency was extremely important for understanding functions and dynamics of miRNA. Herein, an easy-to-obtain DNA-nucleated spherical nucleic acid (DSNA) was constructed by one pair of hairpin probes “sticked” on self-assembled three-dimensional (3D) urchin-like DNA nanostructures (UDNs) to realize sensitive imaging of tumor-related miRNA in living cells. UDNs were prepared through four short single-stranded DNA by a simple “one-pot” annealing process that took only a few minutes. Moreover, sticking the reactants of hairpin probes on the UDNs not only sped up the reaction kinetic rate, but also improved the sensitivity and efficiency of miRNA-triggered hairpin cascade circuits. Compared with traditional AuNP-based spherical nucleic acid, the DSNA possessed superb biocompatibility and stability, excellent cell internalization capability and accessibility, which endowed them tremendous advantages for the monitoring of miRNA in living cells. Therefore, our developed strategy exhibited great potential in sensitive imaging of intracellular miRNA, offering a new avenue for early tumor diagnosis and related biomedical research.

Cascaded, Feedback-Driven, and Spatially Localized Emergence of Constitutional Dynamic Networks Driven by Enzyme-Free Catalytic DNA Circuits.

The enzyme-free catalytic hairpin assembly (CHA) process is introduced as a functional reaction module for guided, high-throughput, emergence, and evolution of constitutional dynamic networks, CDNs, from a set of nucleic acids. The process is applied to assemble networks of variable complexities, functionalities, and spatial confinement, and the systems provide possible mechanistic pathways for the evolution of dynamic networks under prebiotic conditions. Subjecting a set of four or six structurally engineered hairpins to a promoter P1 leads to the CHA-guided emergence of a [2 × 2] CDN or the evolution of a [3 × 3] CDN, respectively. Reacting of a set of branched three-arm DNA-hairpin-functionalized junctions to the promoter strand activates the CHA-induced emergence of a three-dimensional (3D) CDN framework emulating native gene regulatory networks. In addition, activation of a two-layer CHA cascade circuit or a cross-catalytic CHA circuit and cascaded driving feedback-driven evolution of CDNs are demonstrated. Also, subjecting a four-hairpin-modified DNA tetrahedron nanostructure to an auxiliary promoter strand simulates the evolution of a dynamically equilibrated DNA tetrahedron-based CDN that undergoes secondary fueled dynamic reconfiguration. Finally, the effective permeation of DNA tetrahedron structures into cells is utilized to integrate the four-hairpin-functionalized tetrahedron reaction module into cells. The spatially localized miRNA-triggered CHA evolution and reconfiguration of CDNs allowed the logic-gated imaging of intracellular RNAs. Beyond the bioanalytical applications of the systems, the study introduces possible mechanistic pathways for the evolution of functional networks under prebiotic conditions.

Compute-and-Release Logic-Gated DNA Cascade Circuit for Accurate Cancer Cell Imaging.

Accurate identification of cancer cells is an essential prerequisite for cancer diagnosis and subsequent effective curative interventions. The logic-gate-assisted cancer imaging system that allows a comparison of expression levels between biomarkers, rather than just reading biomarkers as inputs, returns a more comprehensive logical output, improving its accuracy for cell identification. To fulfill this key criterion, we develop a compute-and-release logic-gated double-amplified DNA cascade circuit. This novel system, CAR-CHA-HCR, consists of a compute-and-release (CAR) logic gate, a double-amplified DNA cascade circuit (termed CHA-HCR), and a MnO2 nanocarrier. CAR-CHA-HCR, a novel adaptive logic system, is designed to logically output the fluorescence signals after computing the expression levels of intracellular miR-21 and miR-892b. Only when miR-21 is present and its expression level is above the threshold CmiR-21 > CmiR-892b, the CAR-CHA-HCR circuit performs a compute-and-release operation on free miR-21, thereby outputting enhanced fluorescence signals to accurately image positive cells. It is capable of comparing the relative concentrations of two biomarkers while sensing them, thus allowing accurate identification of positive cancer cells, even in mixed cell populations. Such an intelligent system provides an avenue for highly accurate cancer imaging and is potentially envisioned to perform more complex tasks in biomedical studies.

Reliability Analysis of FinFET Based High Performance Circuits

In the VLSI industry, the ability to anticipate variability tolerance is essential to understanding the circuits’ potential future performance. The cadence virtuoso tool is used in this study to assess how PVT fluctuations affect various fin-shaped field effect transistor (FinFET) circuits. In this research, high-performance FinFET-based circuits at 7 nm are discussed with a variation in temperature and voltage. The idea behind the technology is the improvement of power dissipation and delay reduction at the rise of temperature and reduced supply voltage. With the use of a multi-gate predictive model, simulation is carried out employing diverse domino logic at the 7 nm technology node of FinFET files. The proposed set-reset logic circuit and high-speed cascade circuit method shows less power dissipation and delay compared to the existing current mirror footed domino, high-speed clocked delay, and modified high-speed clocked delay with a variation of temperature and supply voltage. For the proposed set-reset logic circuit and high speed cascade circuit, a Monte Carlo simulation is done to find the mean and standard deviation. FinFET simulations are run on the suggested circuit for the reduction of delay for the rise of temperature and reduction of supply voltage from 0.7 V to 0.3 V. In comparison, the proposed method results in a maximum power decrease compared to existing ones. Compared to the existing one, proposed techniques achieve a maximum delay and area reduction.

Self-assembled DNA nanoparticles enable cascade circuits for mRNA detection and imaging in living cells

Fluorescence molecular probes have been regarded as a valuable tool for RNA detection and imaging. However, the pivotal challenge is how to develop an efficient fluorescence imaging platform for accurate identification of RNA molecules with low expression in complicated physiological environments. Herein, we construct the DNA nanoparticles to glutathione (GSH)-responsive controllable release of hairpin reactants for catalytic hairpin assembly (CHA)-hybridization chain reaction (HCR) cascade circuits, which enables the analysis and imaging of low-abundance target mRNA in living cells. The aptamer-tethered DNA nanoparticles are constructed via the self-assembly of single-stranded DNAs (ssDNAs), exhibiting sufficient stability, cell-specific penetration, and precise controllability. Moreover, the in-depth integration of different DNA cascade circuits shows the improved sensing performance of DNA nanoparticles in live cell analysis. Therefore, through the combination of multi-amplifiers and programmable DNA nanostructure, the developed strategy enables accurately triggered release of hairpin reactants and further achieves sensitive imaging and quantitative evaluation of survivin mRNA in carcinoma cells, which provides a potential platform to facilitate RNA fluorescence imaging applications in early clinical cancer theranostics.

Dual-Band Bandstop Filtering Cable Design Using Defected Conductor Layer With Asymmetric Spiral Structure

In this article, we present a novel dual-band bandstop filtering cable by surrounding inner wire with asymmetric bridge-connected spiral-shaped defected structure on outer conductor. The simulation results demonstrate that the proposed dual-band bandstop filtering cable can be modeled by two parallel RLC resonance circuits in cascade and the performance can be easily adjusted by changing the structural parameters. The well-designed dual-band bandstop filtering cable can be fabricated by core wire surrounding with polytetrafluoroethylene (PTFE) dielectric layer and polyimide defected conductor layer with asymmetric bridge-connected spiral-shaped defected structures. The proposed filtering cable with ten asymmetric bridge-connected spiral-shaped defected resonators on defected conductor layer has a compact size of diameter 1.66 mm and length 200 mm, and its insertion loss in experimental results is greater than 20 dB at 0.88 and 1.3 GHz. Compared with the traditional microstrip bandstop filter, the proposed dual-band bandstop filtering cable can achieve better stopband and passband performance within more compact size. In essence, it is a cable replacement technology, which can improve electromagnetic compatibility (EMC) performance at system level and equipment level, and has the potential to be widely used in wireless system.

Circuit network theory of n-horizontal bridge structure

This research investigates a complex n order cascading circuit network with embedded horizontal bridge circuits with the N-RT method. The contents of the study include equivalent resistance analytical formula and complex impedance characteristics of the circuit network. The research idea is as follows. Firstly the equivalent model of n-order resistance network is established, and a fractional difference equation model is derived using Kirchhoff’s law. Secondly, the equivalent transformation method is employed to transform the fractional equation into a simple linear difference equation, and its particular solution is computed. Then the solution to the difference equation is used to derive the effective resistance of the resistance network of the embedded horizontal bridge circuit, and various special cases of equivalent resistance formula are analyzed and the correctness of the analysis model gets verified. Finally, as an expanded application, the equivalent complex impedance of LC network is studied, and Matlab drawing tool is employed to offer the equivalent impedance with various variables of the graph. Our results provide new research ideas and theoretical basis for relevant scientific researches and practical applications.

Construction of Multiple Logic Circuits Based on Allosteric DNAzymes.

In DNA computing, the implementation of complex and stable logic operations in a universal system is a critical challenge. It is necessary to develop a system with complex logic functions based on a simple mechanism. Here, the strategy to control the secondary structure of assembled DNAzymes’ conserved domain is adopted to regulate the activity of DNAzymes and avoid the generation of four-way junctions, and makes it possible to implement basic logic gates and their cascade circuits in the same system. In addition, the purpose of threshold control achieved by the allosteric secondary structure implements a three-input DNA voter with one-vote veto function. The scalability of the system can be remarkably improved by adjusting the threshold to implement a DNA voter with 2n + 1 inputs. The proposed strategy provides a feasible idea for constructing more complex DNA circuits and a highly integrated computing system.

DNA-Templated Bioorthogonal Reactions via Catalytic Hairpin Assembly for Precise RNA Imaging in Live Cells.

There has been a significant interest in developing proximity-induced bioorthogonal reactions for nucleic acid detection and imaging, owing to their high specificity and tunable reaction kinetics. Herein, we reported the first design of a fluorogenic sensor by coupling a bioorthogonal reaction with a DNA cascade circuit for precise RNA imaging in live cells. Two DNA hairpin probes bearing tetrazines or vinyl ether caged fluorophores were designed and synthesized. Upon target mRNA triggering catalytic hairpin assembly, the chemical reaction partners were brought in a spatial proximity to yield high effective concentrations, which dramatically facilitated the bioorthogonal reaction efficiency to unmask the vinyl ether group to activate fluorescence. The proposed fluorogenic sensor was demonstrated to have a high signal-to-noise ratio up to ∼30 fold and enabled the sensitive detection of target mRNA with a detection limit of 4.6 pM. Importantly, the fluorogenic sensor presented low background signals in biological environments due to the unique "click to release" feature, avoiding false positive results caused by unspecific degradation. We also showed that the fluorogenic sensor could accurately image mRNA in live cells and distinguish the relative mRNA expression levels in both tumor and normal cells. Benefiting from these significant advantages, our method provides a useful tool for basic studies of bioorthogonal chemistry and early clinical diagnosis.

Terminal Deoxynucleotidyl Transferase Extension-Dominated In Situ Signal Attenuation-Free Electrochemical Platform and Its Logic Gate Manipulation

Signal generation of traditional electrochemical biosensors suffers from the random diffusion of electroactive probes in a electrolyte solution, which is accompanied by poor reaction kinetics and low signal stability from complex biological systems. Herein, a novel circuit system with autonomous compensation solution ohmic drop (noted as “fast-scan cyclic voltammetry (FSCV)”) is developed to solve the above problems, and employed to achieve terminal deoxynucleotide transferase (TdT) and its small molecule inhibitor analysis. At first, a typical TdT-mediated catalytic polymerization in the conditions of original DNA, deoxythymine triphosphate (dTTP) and Hg2+ is applied for the electrode assembly. The novel electrochemical method can provide some unattenuated signals due to in situ Hg redox reaction, thus improving reaction kinetics and signal stability. This approach is mainly dependent on TdT-mediated reaction, so it can be applied properly for TdT investigation, and a detection limit of 0.067 U ml−1 (S/N = 3) is achieved successfully. More interesting, we also mimic the function of TdT-related signal communication in various logic gates such as YES, NOT, AND, N-IMPLY, and AND-AND-N-IMPLY cascade circuit. This study provides a new method for the detection of TdT biomarkers in many types of diseases and the construction of a signal attenuation-free logic gate.

A Novel Reflection-Type Polarization Convertor Design Using Connected Orthogonal Tightly Coupled Dipole Arrays

Herein, we design a polarization convertor (PC) using two orthogonally polarized, tightly coupled dipole arrays (TCDAs) connected in cascade, of which one is used for the reception of incident waves and the other is used for their transmission. The operation of the PC is elucidated via a cascading equivalent circuit of unit cells of orthogonally polarized TCDAs. The critical role of vertical metal strips (VMSs) with regard to wide-angle insensitivity is discussed. Moreover, it is found that the undesirable common mode occurs under oblique incidence and its frequency is related to the size-to-height ratio of the unit cell. To demonstrate the performance of the proposed PC, a 40 × 40 array structure is fabricated and measured using the Naval Research Laboratory (NRL) arch method. The simulated bandwidth range is 2.06 to 14.54 GHz (7.06:1) for a polarization conversion ratio (PCR) ≥ 80%, and the measured bandwidth range is 1.68 to 14.51 GHz (8.64:1) for a PCR ≥ 72% at an incident angle of 10°. At an incident angle of 40°, the simulated and measured bandwidths are 2.15 to 14.13 GHz (6.57:1) and 2.43 to 13.87 GHz (5.71:1) for a PCR ≥ 80%, respectively.

Real-Time Investigation of Intracellular Polynucleotide Kinase Using a Cascaded Amplification Circuit.

Polynucleotide kinase (PNK) shows an in-depth correlationship with DNA repair and metabolism processes. The in situ visualization of intracellular PNK revealed an extremely biological significance in supplementing reliable and quantitative information on its spatiotemporal distribution in live cells. Herein, we developed a versatile cascaded DNA amplification circuit through the integration of catalytic DNA assembly and hybridization chain reaction circuits and realized the accurate evaluation of intracellular PNK activity via the Förster resonance energy transfer (FRET) principle. Initially, without PNK, trigger T was firmly caged in the PNK-recognizing hairpin HT, resulting in no disturbance of the concatenated circuit. However, with the introduction of PNK, the 5'-OH terminal of PNK-addressing HT was phosphorylated, then the phosphorylated HT could be subsequently digested by λ exonuclease (λ Exo) to produce trigger T of the cascaded DNA circuit. As a result, the integrated circuit was stimulated to produce an amplified FRET signal for quantitatively monitoring the activity of PNK. Due to the λ Exo-specific digestion of 5'-phosphate DNA and the high signal gain of the cascade circuit, our proposed strategy enables the sensitive analysis of PNK activity in vitro and in complex biological samples. Furthermore, our PNK-sensing platform was extensively explored in HeLa cells for realizing reliable intracellular PNK imaging and thus showed high potential in the future diagnosis and treatment of kinase-related diseases.

Analysis and Implementation of a Frequency Control DC–DC Converter for Light Electric Vehicle Applications

In order to realize emission-free solutions and clean transportation alternatives, this paper presents a new DC converter with pulse frequency control for a battery charger in electric vehicles (EVs) or light electric vehicles (LEVs). The circuit configuration includes a resonant tank on the high-voltage side and two variable winding sets on the output side to achieve wide output voltage operation for a universal LEV battery charger. The input terminal of the presented converter is a from DC microgrid with voltage levels of 380, 760, or 1500 V for house, industry plant, or DC transportation vehicle demands, respectively. To reduce voltage stresses on active devices, a cascade circuit structure with less voltage rating on power semiconductors is used on the primary side. Two resonant capacitors were selected on the resonant tank, not only to achieve the two input voltage balance problem but also to realize the resonant operation to control load voltage. By using the variable switching frequency approach to regulate load voltage, active switches are turned on with soft switching operation to improve converter efficiency. In order to achieve wide output voltage capability for universal battery charger demands such as scooters, electric motorbikes, Li-ion e-trikes, golf carts, luxury golf cars, and quad applications, two variable winding sets were selected to have a wide voltage output (50~160 V). Finally, experiments with a 1 kW rated prototype were demonstrated to validate the performance and benefits of presented converter.

DNAzyme cascade circuits in highly integrated DNA nanomachines for sensitive microRNAs imaging in living cells

DNA molecular probes have emerged as powerful tools for fluorescence imaging of microRNAs (miRNAs) in living cells and thus elucidating functions and dynamics of miRNAs. In particular, the highly integrated DNA probes that can be able to address the robustness, sensitivity and consistency issues in a single assay system were highly desired but remained largely unsolved challenge. Herein, we reported for the first time that the development of the novel DNA nanomachines that split-DNAzyme motif was highly integrated in a single DNA triangular prism (DTP) reactor and can undergo target-activated DNAzyme catalytic cascade circuits, allowing amplified sensing and imaging of tumor-related microRNA-21 (miR-21) in living cells. The DNA nanomachines have shown dynamic responses for target miR-21 with excellent sensitivity and selectivity and demonstrated the potential for living cell imaging of miR-21. With the advantages of facile modular design and assembly, high biostability, low cytotoxicity and excellent cellular internalization, the highly integrated DNA nanomachines enabled accurate and effective monitoring of miR-21 expression levels in living cells. Therefore, our developed strategy may afford a reliable and robust nanoplatform for tumor diagnosis and for related biological research.