DNA-functionalized artificial mechanoreceptor for de novo force-responsive signaling.

Synthetic signaling receptors enable programmable cellular responses coupling with customized inputs. However, engineering a designer force-sensing receptor to rewire mechanotransduction remains largely unexplored. Herein, we introduce nongenetically engineered artificial mechanoreceptors (AMRs) capable of reprogramming non-mechanoresponsive receptor tyrosine kinases (RTKs) to sense user-defined force cues, enabling de novo-designed mechanotransduction. AMR is a modular DNA-protein chimera comprising a mechanosensing-and-transmitting DNA nanodevice grafted on natural RTKs via aptameric anchors. AMR senses intercellular tensile force via an allosteric DNA mechano-switch with tunable piconewton-sensitive force tolerance, actuating a force-triggered dynamic DNA assembly to manipulate RTK dimerization and activate intracellular signaling. By swapping the force-reception ligands, we demonstrate the AMR-mediated activation of c-Met, a representative RTK, in response to the cellular tensile forces mediated by cell-adhesion proteins (integrin, E-cadherin) or membrane protein endocytosis (CI-M6PR). Moreover, AMR also allows the reprogramming of FGFR1, another RTK, to customize mechanobiological function, for example, adhesion-mediated neural stem cell maintenance.

Engineering Anti-CRISPR Proteins to Create CRISPR-Cas Protein Switches for Activatable Genome Editing and Viral Protease Detection.

Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR-Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti-CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry-directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease-responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3-fold). These advancements enable specific proteolysis-inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR-Cas tools for controllable gene manipulation and regulation and clinical diagnostics.

Collagen Anchoring Protein-Nucleic Acid Chimeric Probe for In Situ In Vivo Mapping of a Tumor-Specific Protease.

In situ analysis of biomarkers in the tumor microenvironment (TME) is important to reveal their potential roles in tumor progression and early diagnosis of tumors but remains a challenge. In this work, a bottom-up modular assembly strategy was proposed for a multifunctional protein-nucleic chimeric probe (PNCP) for in situ mapping of cancer-specific proteases. PNCP, containing a collagen anchoring module and a target proteolysis-responsive isothermal amplification sensor module, can be anchored in the collagen-rich TME and respond to the target protease in situ and generate amplified signals through rolling cycle amplification of tandem fluorescent RNAs. Taking matrix metalloproteinase 2 (MMP-2), a tumor-associated protease, as the model, the feasibility of PNCP was demonstrated for the in situ detection of MMP-2 activity in 3D tumor spheroids. Moreover, in situ in vivo mapping of MMP-2 activity was also achieved in a metastatic solid tumor model with high sensitivity, providing a useful tool for evaluating tumor metastasis and distinguishing highly aggressive forms of tumors.

A universal orthogonal imaging platform for living-cell RNA detection using fluorogenic RNA aptamers.

MicroRNAs (miRNAs) are crucial regulators of gene expression at the post-transcriptional level, offering valuable insights into disease mechanisms and prospects for targeted therapeutic interventions. Herein, we present a class of miRNA-induced light-up RNA sensors (miLS) that are founded on the toehold mediated principle and employ the fluorogenic RNA aptamers Pepper and Squash as imaging modules. By incorporating a sensor switch to disrupt the stabilizing stem of these aptamers, our design offers enhanced flexibility and convertibility for different target miRNAs and aptamers. These sensors detect multiple miRNA targets (miR-21 and miR-122) with detection limits of 0.48 and 0.2 nM, respectively, while achieving a robust signal-to-noise ratio of up to 44 times. Capitalizing on the distinct fluorescence imaging channels afforded by Pepper-HBC620 (red) and Squash-DFHBI-1T (green), we establish an orthogonal miRNA activation imaging platform, enabling the simultaneous visualization of different intracellular miRNAs in living cells. Our dual-color orthogonal miLS imaging platform provides a powerful tool for sequence-specific miRNA imaging in different cells, opening up new avenues for studying the intricate functions of RNA in living cells.

A collagen-immobilized nanodevice for in situ ratiometric imaging of cancer biomarkers in the tumor microenvironment.

Monitoring the spatiotemporal dynamics of cancer biomarkers within the tumor microenvironment (TME) is critical to understanding their roles in tumorigenesis. Here, we reported a multifunctional fusion protein (collagen-binding domain and duck circovirus tag fused to mCherry, CBD-mCherry-DCV) capable of binding collagen with high affinity and covalently binding specific nucleic acids with exceptional efficiency. We then constructed a chimeric protein-nucleic acid nanodevice (CPNN) using CBD-mCherry-DCV and an aptamer-based sensing module to enable spatially controlled ratiometric imaging of cancer biomarkers in the TME. The collagen-anchoring module CBD-mCherry-DCV allowed specific immobilization of CPNN on 3D multicellular tumor spheroids, enabling the sensing module to achieve "off-on" fluorescence imaging of cancer biomarkers upon specific target recognition by an aptamer. Taking advantage of the constant fluorescence signal of mCherry and the activatable fluorescence response of Cy5 to specific cancer biomarkers, the detection sensitivity and reliability of CPNN were improved by self-calibrating the signal intensity. Specifically, CPNN enabled ratiometric fluorescence imaging of varying concentrations of exogenous PDGF-BB and ATP in tumor spheroids with a high signal-to-background ratio. Furthermore, it allowed the visual monitoring of endogenous PDGF-BB and ATP released from cells. Overall, this study demonstrates the potential of the nanodevice as a versatile approach for the visualization and imaging of cancer biomarkers in the TME.

Programmable Proteolysis-Activated Transcription for Highly Sensitive Ratiometric Electrochemical Detection of Viral Protease.

Viral proteases play a crucial role in viral infection and are regarded as promising targets for antiviral drug development. Consequently, biosensing methods that target viral proteases have contributed to the study of virus-related diseases. This work presents a ratiometric electrochemical sensor that enables highly sensitive detection of viral proteases through the integration of target proteolysis-activated in vitro transcription and the DNA-functionalized electrochemical interface. In particular, each viral protease-mediated proteolysis triggers the transcription of multiple RNA outputs, leading to amplified ratiometric signals on the electrochemical interface. Using the NS3/4A protease of the hepatitis C virus as a model, this method achieves robust and specific NS3/4A protease sensing with sub-femtomolar sensitivity. The feasibility of this sensor was demonstrated by monitoring NS3/4A protease activities in virus-infected cell samples with varying viral loads and post-infection times. This study provides a new approach to analyzing viral proteases and holds the potential for developing direct-acting antivirals and novel therapies for viral infections.

An Extracellular miRNA-Responsive Artificial Receptor via Dynamic DNA Nano-assembly for Biomarker-Driven Therapy.

MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers and therapeutic targets in various diseases. However, there is currently a lack of molecular strategies that can effectively use disease-associated extracellular miRNAs as input signals to drive therapeutic functions. Herein, we present a modular and programmable miRNA-responsive chimeric DNA receptor (miRNA-CDR) capable of biomarker-driven therapy. By grafting a miRNA-responsive DNA nanodevice on a natural membrane receptor via aptamer anchoring, miRNA-CDR can sense extracellular miRNA levels and autonomously induce dimerization-mediated receptor activation via the complementary-mediated strand displacement reaction-induced dynamic DNA assembly. The sequence programmability of miRNA-CDR allows it to sense and respond to a user-defined miRNA with tunable sensitivity. Moreover, the miRNA-CDR is versatile and customizable to reprogram desirable signaling output via adapting a designated receptor, such as MET and FGFR1. Using a mouse model of drug-induced acute liver injury (DILI), we demonstrate the functionality of a designer miRNA-CDR in rewiring the recognition of the DILI-elevated miR-122 to promote MET signaling of hepatocytes for biomarker-driven in situ repair and liver function restoration. Our synthetic miRNA-CDR platform provides a novel molecular device enabling biomarker-driven therapeutic cellular response, potentially paving the way for improving the precision of cell therapy in regenerative medicine.

Engineering fluorescent protein chromophores with an internal reference for high-fidelity ratiometric G4 imaging in living cells.

G-quadruplexes (G4s) are significant nucleic acid secondary structures formed by guanine-rich sequences. Many single-emission G4 fluorescent probes that are lit up by inhibiting intramolecular rotation have been reported. However, they are non-fluorescent unless structurally rigidified, making them sensitive to other intracellular crowding and confinement environments in the cell, like viscosity. Ratiometric measurements provide built-in self-calibration for signal correction, enabling more sensitive and reliable detection. Herein, we structurally modulate green fluorescent protein (GFP)-like chromophores by integrating the imidazolidinone scaffold of the GFP chromophore and coumarin 6H, obtaining a G4 responsive dual-emission chromophore, called NHCouI. The red emission signal of NHCouI can specifically respond to parallel G4s, while its green emission signal is inert and acts as an internal reference signal. NHCouI-G4 complexes feature high fluorescence quantum yield and excellent anti-photobleaching properties. NHCouI can self-calibrate the signal and avoid viscosity disturbances within the range of major subcellular organelles during G4 imaging in living cells. It is also applied to reflect the difference between apoptosis and ferroptosis via tracking G4s. To the best of our knowledge, NHCouI is the first small molecule G4 probe enabled by internal reference correction capability, opening up new avenues for dual-emission chromophore development and high-fidelity and reliable analysis in G4 imaging research.

Emulating Titin by a Multidomain DNA Structure.

Titin, a giant protein containing multiple tandem domains, is essential in maintaining the superior mechanical performance of muscle. The consecutive and reversible unfolding and refolding of the domains are crucial for titin to serve as a modular spring. Since the discovery of the mechanical features of a single titin molecule, the exploration of biomimetic materials with titin-emulating modular structures has been an active field. However, it remains a challenge to prepare these modular polymers on a large scale due to the complex synthesis process. In this study, we propose modular DNA with multiple hairpins (MH-DNA) as the fundamental block for the bottom-up design of advanced materials. By analyzing the unfolding and refolding dynamics of modular hairpins by atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS), we find that MH-DNA shows comparable stability to those of polyproteins like titin. The unique low hysteresis of modular hairpin makes it an ideal molecular spring with remarkable mechanical efficiency. On the basis of the well-established DNA synthesis techniques, we anticipate that MH-DNA can be used as a promising building block for advanced materials with a combination of superior structural stability, considerable extensibility, and high mechanical efficiency.

Multifunctional stimuli-responsive chemogenetic platform for conditional multicolor cell-selective labeling.

Multicolor conditional labeling is a powerful tool that can simultaneously and selectively visualize multiple targets for bioimaging analysis of complex biological processes and cellular features. We herein report a multifunctional stimuli-responsive Fluorescence-Activating and absorption-Shifting Tag (srFAST) chemogenetic platform for multicolor cell-selective labeling. This platform comprises stimuli-responsive fluorogenic ligands and the organelle-localizable FAST. The physicochemical properties of the srFAST ligands can be tailored by modifying the optical-tunable hydroxyl group with diverse reactive groups, and their chemical decaging process caused by cell-specific stimuli induces a conditionally activatable fluorescent labeling upon binding with the FAST. Thus, the resulting switch-on srFASTs were designed for on-demand labeling of cells of interest by spatiotemporally precise photo-stimulation or unique cellular feature-dependent activation, including specific endogenous metabolites or enzyme profiles. Furthermore, diverse enzyme-activatable srFAST ligands with distinct colors were constructed and simultaneously exploited for multicolor cell-selective labeling, which allow discriminating and orthogonal labeling of three different cell types with the same protein tag. Our method provides a promising strategy for designing a stimuli-responsive chemogenetic labeling platform via facile molecular engineering of the synthetic ligands, which has great potential for conditional multicolor cell-selective labeling and cellular heterogeneity evaluation.

Advances in G-quadruplexes-based fluorescent imaging.

G-quadruplexes (G4s), the noncanonical nucleic acid secondary structure, form within guanine-rich DNA or RNA sequences. G4s formation can affect chromatin architecture and gene regulation and has been associated with various cellular functions, including DNA replication, transcription, and genome maintenance. Visualizing and detecting G4s precisely in such processes is essential to increasing our understanding of G4s biology. Considerable attention has focused on the G4s targeting molecular imaging studies. Besides, fluorescent light-up aptamers (FLAPs, also referred to as fluorogenic aptamers) have gained momentum, which commonly have a G4 scaffolding for imaging intracellular RNAs and metabolites. In this review, we first introduce several representative fluorescent imaging approaches for tracking G4s in cells and in vivo. We also discuss the potential of G4-containing FLAPs in bioimaging and summarize current developments in this field from the standpoint of fluorescent molecules. Finally, we discuss the present challenges and future potential of G4 imaging and G4-containing FLAPs development.

Spatially Reprogramed Receptor Organization to Switch Cell Behavior Using a DNA Origami-Templated Aptamer Nanoarray.

Receptor oligomerization is a highly complex molecular process that modulates divergent cell signaling. However, there is a lack of molecular tools for systematically interrogating how receptor oligomerization governs the signaling response. Here, we developed a DNA origami-templated aptamer nanoarray (DOTA) that enables precise programming of the oligomerization of receptor tyrosine kinases (RTK) with defined valency, distribution, and stoichiometry at the ligand-receptor interface. The DOTA allows for advanced receptor manipulations by arraying either monomeric aptamer ligands (mALs) that oligamerize receptor monomers to elicit artificial signaling or dimeric aptamer ligands (dALs) that preorganize the receptor dimer to recapitulate natural activation. We demonstrated that the multivalency and nanoscale spacing of receptor oligomerization coordinately influence the activation level of receptor tyrosine kinase signaling. Furthermore, we illustrated that DOTA-modulated receptor oligomerization could function as a signaling switch to promote the transition from epithelia to mesenchymal-like cells, demonstrating robust control over cellular behaviors. Together, we present a versatile all-in-one DNA nanoplatform for the systematical investigation and regulation of receptor-mediated cellular response.

Study and Classification of Porosity Stress Sensitivity in Shale Gas Reservoirs Based on Experiments and Optimized Support Vector Machine Algorithm for the Silurian Longmaxi Shale in the Southern Sichuan Basin, China.

To understand the characteristics of variation in porosity and permeability, the physical properties of the shale reservoir under different stress conditions play an important role in guiding shale gas production. With the shale of the Wufeng-Longmaxi Formation in the south of the Sichuan Basin as the research object, stress-dependent porosity and permeability test, high-pressure mercury injection, and scanning electron microscope test were performed in this study to thoroughly analyze the variation in physical properties of different shale lithofacies with effective stress. Besides, the stress sensitivity of different lithofacies reservoirs was evaluated by using parameters such as pore compressibility coefficient (PCC) and porosity sensitivity exponent (PSE), while the optimized support vector machine (SVM) algorithm was adopted to predict the coefficient of reservoir porosity sensitivity. According to the research results, the porosity and permeability of shale reservoirs decline as a negative exponential function. When the effective stress falls below 15 MPa, the damage rate of permeability/porosity increases rapidly with the rise of effective stress. By contrast, the permeability curvature of the shale reservoirs plunges with the rise of effective stress. It was discovered that a higher siliceous content results in a higher permeability curvature of shale, indicating the greater stress sensitivity of the reservoir. The ratio of matrix porosity to microfracture porosity determines the PSE, which is relatively low, and low aspect ratio pores contribute to high porosity compressibility and stress sensitivity. Young's modulus shows a negative correlation with pore compressibility and a positive correlation with Poisson's ratio. High clay minerals have a large number of low aspect ratio pores and a low elastic modulus, which leads to both high PCC and low PSE. Based on the principal component analysis, a multiclassification SVM model was established to predict the PSE, revealing that the accuracy of the sigmoid, radial basis function (RBF), and linear kernel function is consistently above 70%. According to error analysis, the accuracy can exceed 80% with the RBF kernel function and appropriate penalty factor. The research results serve to advance the research on the parameters related to overburden pressure, porosity, and permeability. Moreover, the optimized SVM algorithm is applied to make a classification prediction, which provides a reference for shale reservoir exploration and development both in theory and practice.

Visualization of Deep Tissue G-quadruplexes with a Novel Large Stokes-Shifted Red Fluorescent Benzothiazole Derivative.

G-quadruplex (G4) is a noncanonical nucleic acid secondary structure that has implications for various physiological and pathological processes and is thus essential to exploring new approaches to G4 detection in live cells. However, the deficiency of molecular imaging tools makes it challenging to visualize the G4 in ex vivo tissue samples. In this study, we established a G4 probe design strategy and presented a red fluorescent benzothiazole derivative, ThT-NA, to detect and image G4 structures in living cells and tissue samples. By enhancing the electron-donating group of thioflavin T (ThT) and optimizing molecular structure, ThT-NA shows excellent photophysical properties, including red emission (610 nm), a large Stokes shift (>100 nm), high sensitivity selectivity toward G4s (1600-fold fluorescence turn-on ratio) and robust two-photon fluorescence emission. Therefore, these features enable ThT-NA to reveal the endogenous RNA G4 distribution in living cells and differentiate the cell cycle by monitoring the changes of RNA G4 folding. Significantly, to the best of our knowledge, ThT-NA is the first benzothiazole-derived G4 probe that has been developed for imaging G4s in ex vivo cancer tissue samples by two-photon microscopy techniques.

Kinetics Accelerated CRISPR-Cas12a Enabling Live-Cell Monitoring of Mn2+ Homeostasis.

The CRISPR/Cas12a system has been repurposed as a versatile nuclei acid bio-imaging tool, but its utility in sensing non-nucleic acid analytes in living cells has been less exploited. Herein, we demonstrated the ability of Mn2+ to accelerate cleavage kinetics of Cas12a and deployed for live-cell Mn2+ sensing by leveraging the accelerated trans-cleavage for signal reporting. In this work, we found that Mn2+ could significantly boost both the cis-cleavage and trans-cleavage activities of Cas12a. On the basis of this phenomenon, we harnessed CRISPR-Cas12a as a direct sensing system for Mn2+, which achieved robust Mn2+ detection in the concentration range of 0.5-700 µM within 15 min in complex biological samples. Furthermore, we also demonstrated the versatility of this system to sense Mn2+ in the cytoplasm of living cells. With the usage of a conditional guide RNA, this Cas12a-based sensing method was applied to study the cytotoxicity of Mn2+ in living nerve cells, offering a valuable tool to reveal the cellular response of nerve cells to Mn2+ disorder and homeostasis.

A switchable Cas12a enabling CRISPR-based direct histone deacetylase activity detection.

The efficient and robust signal reporting ability of CRISPR-Cas system exhibits huge value in biosensing, but its applicability for non-nucleic acid analyte detection relies on the coupling of additional recognition modules. To address this limitation, we described a switchable Cas12a and exploited it for CRISPR-based direct analysis of histone deacetylase (HDAC) activity. Starting from the acetylation-mediated inactivation of Cas12a by anti-CRISPR protein AcrVA5, we demonstrated that the acetyl-inactivated Cas12a could be reversibly activated by HDAC-mediated deacetylation based on computational simulations (e.g., deep learning and protein-protein docking analysis) and experimental verifications. By leveraging this switchable Cas12a for both target sensing and signal amplification, we established a sensitive one-pot assay capable of detecting deacetylase sirtuin-1 with sub-nanomolar sensitivity, which is 50 times lower than the standard two-step peptide-based assay. The versability of this assay was validated by the sensitive assessment of cellular HDAC activities in different cell lines with good accuracy, making it a valuable tool for biochemical studies and clinical diagnostics.

A Chimeric Conjugate of Antibody and Programmable DNA Nanoassembly Smartly Activates T Cells for Precise Cancer Cell Targeting.

Synthetically directing T-cells against tumors emerges as a promising strategy in immunotherapy, while it remains challenging to smartly engage T cells with tunable immune response. Herein, we report an intelligent molecular platform to engineer T-cell recognition for selective activation to potently kill cancer cells. To this end, we fabricated a hybrid conjugate that uses a click-type DNA-protein conjugation to equip the T cell-engaging antibody with two distinct programmable DNA nanoassemblies. By integrating multiple aptameric antigen-recognitions within a dynamic DNA circuit, we achieved combinatorial recognition of triple-antigens on cancer cells for selective T-cell activation after high-order logic operation. Moreover, by coupling a DNA nanostructure, we precisely defined the valence of the antigen-binding aptamers to tune avidity, realizing effective tumor elimination in vitro and in vivo. Together, we present a versatile and programmable strategy for synthetic immunotherapy.

Advances in Designer DNA Nanorobots Enabling Programmable Functions.

The advent of DNA nanotechnology has paved the way for the development of nanoscale robotics capable of executing smart and sophisticated tasks in a programmed and automatic manner. The programmability and customizable functionality of designer DNA nanorobots interfacing with biology would offer great potential for basic and applied research in the interdisciplinary fields of chemistry, biology, and medicine. This review aims to summarize the latest progress in designer DNA nanorobotics enabling programmable functions. We first describe the state-of-art engineering principles and the functional modules used in the rational design of a dynamic DNA nanorobot. Subsequently, we summarize the distinct types of DNA nanorobots performing sensing tasks, sensing-and-actuation, or continuous actuation, highlighting the versatility of designer DNA nanorobots in accurate biosensing, targeted drug delivery, and autonomous molecular operations to promote desired cellular behavior. Finally, we discuss the challenges and opportunities in the development of functional DNA nanorobotics for biomedical applications. We envision that significant progress in DNA-enabled nanorobotics with programmable functions will improve precision medicine in the future.

PAM-less conditional DNA substrates leverage trans-cleavage of CRISPR-Cas12a for versatile live-cell biosensing.

The CRISPR-Cas system has been repurposed as a powerful live-cell imaging tool, but its utility is limited to genomic loci and mRNA imaging in living cells. Here, we demonstrated the potential of the CRISPR-Cas system as a generalizable live-cell biosensing tool by extending its applicability to monitor diverse intracellular biomolecules. In this work, we engineered a CRISPR-Cas12a system with a generalized stimulus-responsive switch mechanism based on PAM-less conditional DNA substrates (pcDNAs). The pcDNAs with stimulus-responsiveness toward a trigger were constructed from the DNA substrates featuring no requirement of a protospacer-adjacent motif (PAM) and a bubble structure. With further leveraging the trans-cleavage activity of CRISPR-Cas12a for signal reporting, we established a versatile CRISPR-based live-cell biosensing system. This system enabled the sensitive sensing of various intracellular biomolecules, such as telomerase, ATP, and microRNA-21, making it a helpful tool for basic biochemical research and disease diagnostics.

A Biomimetic Approach for Spatially Controlled Cell Membrane Engineering Using Fusogenic Spherical Nucleic Acid.

Engineering of the cell plasma membrane using functional DNA is important for studying and controlling cellular behaviors. However, most efforts to apply artificial DNA interactions on cells are limited to external membrane surface due to the lack of suitable synthetic tools to engineer the intracellular side, which impedes many applications in cell biology. Inspired by the natural extracellular vesicle-cell fusion process, we have developed a fusogenic spherical nucleic acid construct to realize robust DNA functionalization on both external and internal cell surfaces via liposome fusion-based transport (LiFT) strategy, which enables applications including the construction of heterotypic cell assembly for programmed signaling pathway and detection of intracellular metabolites. This approach can engineer cell membranes in a highly efficient and spatially controlled manner, allowing one to build anisotropic membrane structures with two orthogonal DNA functionalities.