Identification of the Binding Site between Aptamer sgc8c and PTK7.

Aptamers are single-stranded RNA or DNA molecules that can specifically bind to targets and have found broad applications in cancer early-stage detection, accurate drug delivery, and precise treatment. Although various aptamer screening methods have been developed over the past several decades, the accurate binding site between the target and the aptamer cannot be characterized during a typical aptamer screening process. In this research, we chose a widely used aptamer screened by our group, sgc8c, and its target protein tyrosine kinase 7 (PTK7) as the model aptamer and target and tried to determine the binding site between aptamer sgc8c and PTK7. Through sequential protein truncation, we confirmed that the exact binding site of sgc8c was within the region of Ig 3 to Ig 4 in the extracellular domain of PTK7. Using in vitro expressed Ig (3-4), we successfully acquired the crystal of an sgc8c-Ig (3-4) binding complex. The possible sgc8c-binding amino acid residues on PTK7 and PTK7-binding nucleotide residues on sgc8c were further identified and simulated by mass spectrometry and molecular dynamics simulation and finally verified by aptamer/protein truncation and mutation.

DNA-Based Molecular Machines: Controlling Mechanisms and Biosensing Applications.

The rise of DNA nanotechnology has driven the development of DNA-based molecular machines, which are capable of performing specific operations and tasks at the nanoscale. Benefitting from the programmability of DNA molecules and the predictability of DNA hybridization and strand displacement, DNA-based molecular machines can be designed with various structures and dynamic behaviors and have been implemented for wide applications in the field of biosensing due to their unique advantages. This review summarizes the reported controlling mechanisms of DNA-based molecular machines and introduces biosensing applications of DNA-based molecular machines in amplified detection, multiplex detection, real-time monitoring, spatial recognition detection, and single-molecule detection of biomarkers. The challenges and future directions of DNA-based molecular machines in biosensing are also discussed.

Dual-responsive 3D DNA nanomachines cascaded hybridization chain reactions for novel self-powered flexible microRNA-detecting platform.

The microRNA-21 is closely related to chromatin remodeling and epigenetic regulation. In this work, an efficient double-response 3D DNA nanomachine (DRDN) was assembled by co-immobilizing two different lengths of hairpin DNA on the surface of gold nanoparticles (AuNPs) to capture microRNA-21 (miRNA-21), recycle miRNA-21, and trigger hybridization chain reactions (HCR). This work reports the fabrication of a laser-scribed graphene (LSG) electrode with excellent flexibility and electrical conductivity by laser-scribing commercial polyimide films (PI). The as-proposed self-powered biosensing platform presents significantly increased instantaneous current to in real-time monitor miRNA-21 by a capacitor. The biosensing platform exhibited highly sensitive detection of miRNA-21 with a detection limit of 0.142 fM in the range of 0.5 fM to 1 × 104 fM, and demonstrated high efficiency in the analysis of the tumor markers.

Self-Powered Biosensor Based on DNA Walkers for Ultrasensitive MicroRNA Detection.

A novel self-powered biosensor is fabricated for ultrasensitive microRNA-21 (miRNA-21) detection, which includes an enzymatic biofuel cell (EBFC), DNA walkers, a digital multimeter (DMM), and a capacitor. As a novel strategy for signal amplification, DNA walkers are designed in the cathode, while the capacitor stores electrochemical energy from the EBFC to further boost the instantaneous current displayed by the DMM. When miRNA-21 is present, the DNA walkers are provoked to walk from as-opened hairpin structures to other hairpin structures, generating double-strand DNA structures, which stimulate [Ru(NH3)6]3+ to be adsorbed on the cathode surface by electrostatic interaction. Afterward, [Ru(NH3)6]3+ is reduced to [Ru(NH3)6]2+, and the open circuit voltage (EOCV) is significantly increased. Depending on the approach of signal amplification from DNA walkers, this biosensor displays an ultrasensitive assay toward miRNA-21 in the range of 0.5 to 104 fM, with a detection limit of 0.15 fM. In addition, this self-powered biosensor displays high selectivity for miRNA-21 assay in human serum samples.

Phase Separation of DNA-Encoded Artificial Cells Boosts Signal Amplification for Biosensing.

Life-like hierarchical architecture shows great potential for advancing intelligent biosensing, but modular expansion of its sensitivity and functionality remains a challenge. Drawing inspiration from intracellular liquid-liquid phase separation, we discovered that a DNA-encoded artificial cell with a liquid core (LAC) can enhance peroxidase-like activity of Hemin and its DNA G-quadruplex aptamer complex (DGAH) without substrate-selectivity, unlike its gelled core (GAC) counterpart. The LAC is easily engineered as an ultrasensitive biosensing system, benefiting from DNA's high programmability and unique signal amplification capability mediated by liquid-liquid phase separation. As proof of concept, its versatility was successfully demonstrated by coupling with two molecular recognition elements to monitor tumor-related microRNA and profile cancer cell phenotypes. This scalable design philosophy offers new insights into the design of next generation of artificial cells-based biosensors.

Membrane-anchored DNA nanojunctions enable closer antigen-presenting cell-T-cell contact in elevated T-cell receptor triggering.

How the engagement of a T-cell receptor to antigenic peptide-loaded major histocompatibility complex on antigen-presenting cells (APCs) initiates intracellular signalling cascades in T cells is not well understood. In particular, the dimension of the cellular contact zone is regarded as a determinant, but its influence remains controversial. This is due to the need for appropriate strategies for manipulating intermembrane spacing between the APC-T-cell interfaces without involving protein modification. Here we describe a membrane-anchored DNA nanojunction with distinct sizes to extend, maintain and shorten the APC-T-cell interface down to 10 nm. Our results suggest that the axial distance of the contact zone is critical in T-cell activation, presumably by modulating protein reorganization and mechanical force. Notably, we observe the promotion of T-cell signalling by shortening the intermembrane distance.

A Dynamic Control Center Based on a DNA Reaction Network for Programmable Building of DNA Nanostructures.

DNA-based nanostructures allow for complex self-assembly with nanometer precision through the specificity of Watson-Crick base pairing, but network behavior-directed control of the kinetic process is less studied. Here we show how the DNA reaction network (DRN), which has emerged as a reliable and programmable way to implement artificial network dynamics, can be built as the control center of programmable nanostructures, allowing spatiotemporal control over the dynamic behavior of DNA nanotubes. We chose a common network motif in biological control systems, the feed-forward loop, as the model network and demonstrated that dynamic behaviors, such as self-tuning control and multilayer hierarchical assembly, could be programmed by constructing an inhibition network and an excitation network, separately, in buffer solution and inside protocells.

Robust Covalent Aptamer Strategy Enables Sensitive Detection and Enhanced Inhibition of SARS-CoV-2 Proteins.

Aptamer-based detection and therapy have made substantial progress with cost control and easy modification. However, the conformation lability of an aptamer typically causes the dissociation of aptamer-target complexes during harsh washes and other environmental stresses, resulting in only moderate detection sensitivity and a decreasing therapeutic effect. Herein, we report a robust covalent aptamer strategy to sensitively detect nucleocapsid protein and potently neutralize spike protein receptor binding domain (RBD), two of the most important proteins of SARS-CoV-2, after testing different cross-link electrophilic groups via integrating the specificity and efficiency. Covalent aptamers can specifically convert aptamer-protein complexes from the dynamic equilibrium state to stable and irreversible covalent complexes even in harsh environments. Covalent aptamer-based ELISA detection of nucleocapsid protein can surpass the gold standard, antibody-based sandwich ELISA. Further, covalent aptamer performs enhanced functional inhibition to RBD protein even in a blood vessel-mimicking flowing circulation system. The robust covalent aptamer-based strategy is expected to inspire more applications in accurate molecular modification, disease biomarker discovery, and other theranostic fields.

Ligand Dilution Analysis Facilitates Aptamer Binding Characterization at the Single-Molecule Level.

Cell-specific aptamers offer a powerful tool to study membrane receptors at the single-molecule level. Most target receptors of aptamers are highly expressed on the cell surface, but difficult to analyze in situ because of dense distribution and fast velocity. Therefore, we herein propose a random sampling-based analysis strategy termed ligand dilution analysis (LDA) for easily implemented aptamer-based receptor study. Receptor density on the cell surface can be calculated based on a regression model. By using a synergistic ligand dilution design, colocalization and differentiation of aptamer and monoclonal antibody (mAb) binding on a single receptor can be realized. Once this is accomplished, precise binding site and detailed aptamer-receptor binding mode can be further determined using molecular docking and molecular dynamics simulation. The ligand dilution strategy also sets the stage for an aptamer-based dynamics analysis of two- and three-dimensional motion and fluctuation of highly expressed receptors on the live cell membrane.

Network topology-directed design of molecular CPU for cell-like dynamic information processing.

Natural cells (NCs) can automatically and continuously respond to fluctuant external information and distinguish meaningful stimuli from weak noise depending on their powerful genetic and protein networks. We herein report a network topology-directed design of dynamic molecular processing system (DMPS) as a molecular central processing unit that powers an artificial cell (AC) able to process fluctuant information in its immediate environment similar to NCs. By constructing a mixed cell community, ACs and NCs have synchronous response to fluctuant extracellular stimuli under physiological condition and in a blood vessel-mimic circulation system. We also show that fluctuant bioinformation released by NCs can be received and processed by ACs. The molecular design of DMPS-powered AC is expected to allow a profound understanding of biological systems, advance the construction of intelligent molecular systems, and promote more elegant bioengineering applications.

Ratiometric afterglow luminescent nanoplatform enables reliable quantification and molecular imaging.

Afterglow luminescence is an internal luminescence pathway that occurs after photo-excitation, holds great promise for non-background molecular imaging in vivo, but suffer from poor quantitative ability owing to luminescent attenuation over time. Moreover, the inert structure and insufficient reactive sites of current afterglow materials make it hard to design activatable afterglow probes for specific detection. Here, we report a ratiometric afterglow luminescent nanoplatform to customize various activatable afterglow probes for reliable quantification and molecular imaging of specific analytes, such as NO, ONOO- or pH. Notably, these afterglow probes can not only address the attenuation of afterglow intensity and eliminate the interference of factors (e.g., laser power, irradiation time, and exposure time), but also significantly improve the imaging reliability in vivo and signal-to-background ratios (~1200-fold), both of which enable more reliable quantitative analysis in biological systems. Moreover, as a proof-of-concept, we successfully design an NO-responsive ratiometric afterglow nanoprobe, RAN1. This nanoprobe can monitor the fluctuations of intratumoral NO, as a biomarker of macrophage polarization, making it possible to real-time dynamically evaluate the degree cancer immunotherapy, which provides a reliable parameter to predict the immunotherapeutic effect.

ResNet-BiLSTM: A Multiscale Deep Learning Model for Heartbeat Detection Using Ballistocardiogram Signals.

As the heartbeat detection from ballistocardiogram (BCG) signals using force sensors is interfered by respiratory effort and artifact motion, advanced signal processing algorithms are required to detect the J-peak of each BCG signal so that beat-to-beat interval can be identified. However, existing methods generally rely on rule-based detection of a fixed size, without considering the rhythm features in a large time scale covering multiple BCG signals. Methods. This paper develops a deep learning framework based on ResNet and bidirectional long short-term memory (BiLSTM) to conduct beat-to-beat detection of BCG signals. Unlike the existing methods, the proposed network takes multiscale features of BCG signals as the input and, thus, can enjoy the complementary advantages of both morphological features of one BCG signal and rhythm features of multiple BCG signals. Different time scales of multiscale features for the proposed model are validated and analyzed through experiments. Results. The BCG signals recorded from 21 healthy subjects are conducted to verify the performance of the proposed heartbeat detection scheme using leave-one-out cross-validation. The impact of different time scales on the detection performance and the performance of the proposed model for different sleep postures are examined. Numerical results demonstrate that the proposed multiscale model performs robust to sleep postures and achieves an averaged absolute error (E abs) and an averaged relative error (E rel) of the heartbeat interval relative to the R-R interval of 9.92 ms and 2.67 ms, respectively, which are superior to those of the state-of-the-art detection protocol. Conclusion. In this work, a multiscale deep-learning model for heartbeat detection using BCG signals is designed. We demonstrate through the experiment that the detection with multiscale features of BCG signals can provide a superior performance to the existing works. Further study will examine the ultimate performance of the multiscale model in practical scenarios, i.e., detection for patients suffering from cardiovascular disorders with night-sleep monitoring.

A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging.

Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

DNA-based artificial molecular signaling system that mimics basic elements of reception and response.

In order to maintain tissue homeostasis, cells communicate with the outside environment by receiving molecular signals, transmitting them, and responding accordingly with signaling pathways. Thus, one key challenge in engineering molecular signaling systems involves the design and construction of different modules into a rationally integrated system that mimics the cascade of molecular events. Herein, we rationally design a DNA-based artificial molecular signaling system that uses the confined microenvironment of a giant vesicle, derived from a living cell. This system consists of two main components. First, we build an adenosine triphosphate (ATP)-driven DNA nanogatekeeper. Second, we encapsulate a signaling network in the biomimetic vesicle, consisting of distinct modules, able to sequentially initiate a series of downstream reactions playing the roles of reception, transduction and response. Operationally, in the presence of ATP, nanogatekeeper switches from the closed to open state. The open state then triggers the sequential activation of confined downstream signaling modules.

"Apollo Program" in Nanoscale: Landing and Exploring Cell-Surface with DNA Nanotechnology.

Plasma membranes are the fundamental mediators through which cells communicate with their surrounding environment. The techniques to monitor or synthetically manipulate the cell membranes are attractive tools to engineer the functions of cells as well as their local microenvironment. Current advances of biomolecular science enable the insertion of functional compounds onto cell-surface via external integration or genetic engineering to manipulate cell membrane function. Recently, the DNA nanotechnology made it possible to use synthetic DNA as an emerging and promising molecular toolkit for anchoring and exploring cell-surface. In this review, the latest advances of DNA nanotechnology on cell-surface are summarized. We first give an overview of commonly used strategies for installing DNA nanodevices onto cell-surface including amphiphilic interaction, covalent modification, and affinity labeling. Then the biological applications of DNA nanodevices on cell membranes are reviewed. By integrating functional nucleic acids as recognition elements, DNA sensors are fabricated to monitor the cellular microenvironment and membrane activities. In addition, the programmable behaviors of DNA on cell-surface are also discussed, which include biomimicry and the regulation of membrane functions. Finally, we analyze the current challenges in the development of DNA nanotechnology on cell-surface as well as their prospects in bioimaging and cancer therapy.

A psychological health support scheme for medical teams in COVID-19 outbreak and its effectiveness.

BACKGROUND: Medical staff fighting the COVID-19 pandemic are experiencing stress from high occupational risk, panic in the community and the extreme workload. Maintaining the psychological health of a medical team is essential for efficient functioning, but psychological intervention models for emergency medical teams are rare. AIMS: To design a systematic, full-coverage psychological health support scheme for medical teams serving large-scale emergent situations, and demonstrate its effectiveness in a real-world study in Leishenshan Hospital during the COVID-19 epidemic in Wuhan, China. METHODS: The scheme integrates onsite and online mental health resources and features team-based psychosocial support and evidence-based interventions. It contained five modules, including a daily measurement of mood, a daily mood broadcast that promotes positive affirmation, a daily online peer-group activity with themes based on the challenges reported by the team, Balint groups and an after-work support team. The daily mood measurement provides information to the other modules. The scheme also respects the special psychological characteristics of medical staff by promoting their strengths. RESULTS: The scheme economically supported a special medical team of 156 members with only one onsite psychiatrist. Our data reflected that the entire medical team maintained an overall positive outlook (7-9 out of 10 in a Daily Mood Index, DMI) for nearly 6 weeks of continuous working. Since the scheme promoted self-strengths and positive self-affirmation, the number of self-reports of life-related gains were high and played a significant effect on the DMI. Our follow-up investigations also revealed that multiple modules of the scheme received high attention and evaluation levels. CONCLUSION: Our quantitative data from Leishenshan hospital, Wuhan, China, show that the programme is adequate to support the continuous high workload of medical teams. This scheme could be applied to medical teams dealing with emergent situations.

Generating Giant Membrane Vesicles from Live Cells with Preserved Cellular Properties.

Biomimetic giant membrane vesicles, with size and lipid compositions comparable to cells, have been recognized as an attractive experimental alternative to living systems. Due to the similarity of their membrane structure to that of body cells, cell-derived giant plasma membrane vesicles have been used as a membrane model for studying lipid/protein behavior of plasma membranes. However, further application of biomimetic giant membrane vesicles has been hampered by the side-effects of chemical vesiculants and the utilization of osmotic buffer. We herein develop a facile strategy to derive giant membrane vesicles (GMVs) from mammalian cells in biofriendly medium with high yields. These GMVs preserve membrane properties and adaptability for surface modification and encapsulation of exogenous molecules, which would facilitate their potential biological applications. Moreover, by loading GMVs with therapeutic drugs, GMVs could be employed for drug transport to tumor cells, which represents another step forward in the biomedical application of giant membrane vesicles. This study highlights biocompatible GMVs with biomimicking membrane surface properties and adaptability as an ideal platform for drug delivery strategies with potential clinical applications.

Free-standing 2D nanorafts by assembly of 1D nanorods for biomolecule sensing.

Novel materials from self-assembled nanocrystals hold great promise for applications ranging from inorganic catalysis to bio-imaging. However, because of the inherent anisotropic properties, it is challenging to assemble one-dimensional (1D) nanorods into higher-order structures (e.g. 2D sheets or 3D networks) without any support. Here, we have developed a facile strategy for the direct self-assembly of 1D nanorods into free-standing 2D nanorafts with lateral dimensions up to several micrometers. As a general approach, 2D nanorafts with diverse compositions, e.g. MgF2, WO2, CdS, ZnS, and ZnSe nanorafts, have been fabricated from the assembly of their 1D building blocks. More importantly, these nanorafts show high stability even when dispersed in different solvents, making them suitable for various applications. Because of their high porosity and strong adsorption capability, MgF2 nanorafts were investigated to illustrate the collective advantages generated from the assembly platform. Moreover, flexibility in the composition and structure of the building blocks demonstrated in this work will lead to next generation materials with rich functionalities.