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.

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.

Scan and Unlock: A Programmable DNA Molecular Automaton for Cell-Selective Activation of Ligand-Based Signaling.

Selective modulation of ligand-receptor interaction is essential in targeted therapy. In this study, we design an intelligent "scan and unlock" DNA automaton (SUDA) system to equip a native protein-ligand with cell-identity recognition and receptor-mediated signaling in a cell-type-specific manner. Using embedded DNA-based chemical reaction networks (CRNs) on the cell surface, SUDA scans and evaluates molecular profiles of cell-surface proteins via Boolean logic circuits. Therefore, it achieves cell-specific signal modulation by quickly unlocking the protein-ligand in proximity to the target cell-surface to activate its cognate receptor. As a proof of concept, we non-genetically engineered hepatic growth factor (HGF) with distinct logic SUDAs to elicit target cell-specific HGF signaling and wound healing behaviors in multiple heterogeneous cell types. Furthermore, the versatility of the SUDA strategy was shown by engineering tumor necrotic factor-α (TNFα) to induce programmed cell death of target cell subpopulations through cell-specific modulation of TNFR1 signaling.

Titanium Carbide MXenes Mediated In Situ Reduction Allows Label-Free and Visualized Nanoplasmonic Sensing of Silver Ions.

The label-free assay has drawn extensive attention because it does not require a labeling step and enables direct interaction and signal transduction between the sensing unit and target analytes. Herein, we demonstrate a proof-of-principle concept of a label-free and visualized nanoplasmonic strategy for silver ions sensing, where only Ti3C2 MXenes are employed by exploring their excellent adsorption affinity and reductive property toward metal ions. Ag+ was adsorbed onto the surface of Ti3C2 MXene nanosheets, followed by the Ti3C2 MXenes mediated in situ silver nanoparticles (Ag NPs) generation without adding any extra stabilizing or reducing agent. The excellent localized surface plasmon resonances at a particular wavelength provide Ag NPs the capability for colorimetric assay with a detection limit of 0.615 µM. With the assistance of a smartphone, RGB analysis exhibited visualized results consistent with the results measured on a UV-vis spectrometer, promising a budget, simple-operating on-site detection. Moreover, the detection of Ag+ in real samples was achieved with satisfactory results meeting the analysis demand for the Drinking Water Standards of the World Health Organization (WHO) and the United States Environmental Protection Agency (U.S. EPA). These results reveal that Ti3C2 MXenes possess great potential in building convenient label-free colorimetry nanoplatforms and may evoke more inspirations to explore strategies for the direct sensing of analytes.

Target-activated transcription for the amplified sensing of protease biomarkers.

Signal amplification is an effective way to achieve sensitive analysis of biomarkers, exhibiting great promise in biomedical research and clinical diagnosis. Inspired by the transcription process, here we present a versatile strategy that enables effective amplification of proteolysis into nucleic acid signal outputs in a homogeneous system. In this strategy, a protease-activatable T7 RNA polymerase is engineered as the signal amplifier and achieves 3 orders of magnitude amplification in signal gain. The versatility of this strategy has been demonstrated by the development of sensitive and selective assays for protease biomarkers, such as matrix metalloproteinase-2 (MMP-2) and thrombin, with sub-picomole sensitivity, which is 4.3 × 103-fold lower than that of the standard peptide-based method. Moreover, the proposed assay has been further applied in the detection of MMP-2 secreted by cancer cells, as well as in the assessment of MMP-2 levels in osteosarcoma tissue samples, providing a general approach for the monitoring of protease biomarkers in clinical diagnosis.

Biomineralization synthesis of a near-infrared fluorescent nanoprobe for direct glucose sensing in whole blood.

A near-infrared (NIR) fluorescent nanoprobe that enables to circumvent the interference of background absorption and fluorescence in whole blood was developed for the direct sensing of blood glucose. Here, NIR fluorescent protein (iRFP) and glucose oxidase (GOx) were collectively deployed as the templates for the biomineralization of Mn2+ to prepare a NIR fluorescent nanoprobe (iRFP-GOx-MnO2 nanoparticles, iRGMs), in which the fluorescence of iRFP was effectively quenched by MnO2via energy transfer. When the iRGMs were mixed with whole blood samples, GOx can convert blood glucose into gluconic acid, as well as H2O2, which will reduce MnO2 and decompose the iRGMs. As a result, the NIR fluorescence of iRFPs was restored, providing a fluorometric assay for the direct detection of blood glucose. Owing to the high efficiency of the cascade reaction and the low background interference of the NIR fluorescence signal, accurate and rapid analysis of the glucose levels in whole blood samples was achieved using the iRGMs. Moreover, an iRGM-based paper device that only requires 5 microliters of samples was also demonstrated in the direct assay of blood glucose without any pretreatment, affording an alternative approach for the accurate monitoring of blood glucose levels.

Click-Type Protein-DNA Conjugation for Mn2+ Imaging in Living Cells.

A click-type protein-DNA conjugation, named as MnDDC (Mn2+-activated DCV-DNA conjunction), is presented, where DCV (rep protein of duck circovirus) and its target DNA work as the modular blocks to rapidly and effectively generate Mn2+-dependent and site-specific protein-DNA linkage. On the basis of MnDCC, a fluorescent Mn2+ biosensor composed of DCV and a molecular beacon, was developed for rapid sensing of Mn2+ within 2 min with nanomolar sensitivity. Using the proposed biosensor, not only analysis of Mn2+ in real samples (e.g., serum and food), but also wash-free fluorescent imaging of Mn2+ in extracellular environment and cytoplasm have been achieved. Moreover, the MnDDC-based sensor was proved to be a powerful tool for visualization of Mn2+ during exploration of the associated cytotoxicity in living neural cells, which is helpful to reveal the cellular responses toward the disordered homeostasis of Mn2+ in both extracellular and intracellular microenvironments.

Engineering of Nucleic Acids and Synthetic Cofactors as Holo Sensors for Probing Signaling Molecules in the Cellular Membrane Microenvironment.

The comprehensive understanding of the mechanisms underlying the interaction of cells with their membrane microenvironment is of great value for fundamental biological research; however, tracking biomolecules on cell surfaces with high temporal and spatial resolution remains a challenge. Herein, a modular strategy is presented for the construction of cell surface DNA-based sensors by engineering DNA motifs and synthetic cofactors. In this strategy, a stimuli-reactive organic molecule is employed as the cofactor for the DNA motif, and the self-assembly of them forms a FRET-based holo DNA-based sensor. With the use of the DNA-based sensors, the versatility of this modular strategy has been demonstrated in the ratiometric imaging of the cellular extrusion process of endogenous signaling molecules, including sulfur dioxide derivatives and nitric oxide.

Genome-wide Identification and Characterization of Enhancers Across 10 Human Tissues.

Background: Enhancers can act as cis-regulatory elements (CREs) to control development and cellular function by regulating gene expression in a tissue-specific and ubiquitous manner. However, the regulatory network and characteristic of different types of enhancers (e.g., transcribed/non-transcribed enhancers, tissue-specific/ubiquitous enhancers) across multiple tissues are still unclear. Results: Here, a total of 53,924 active enhancers and 10,307 enhancer-associated RNAs (eRNAs) in 10 tissues (adrenal, brain, breast, heart, liver, lung, ovary, placenta, skeletal muscle and kidney) were identified through the integration of histone modifications (H3K4me1, H3K27ac and H3K4me3) and DNase I hypersensitive sites (DHSs) data. Moreover, 40,101 tissue-specific enhancers (TS-Enh), 1,241 ubiquitously expressed enhancers (UE-Enh) as well as transcribed enhancers (T-Enh), including 7,727 unidirectionally transcribed enhancers (1D-Enh) and 1,215 bidirectionally transcribed enhancers (2D-Enh) were defined in 10 tissues. The results show that enhancers exhibited high GC content, genomic variants and transcription factor binding sites (TFBS) enrichment in all tissues. These characteristics were significantly different between TS-Enh and UE-Enh, T-Enh and NT-Enh, 2D-Enh and 1D-Enh. Furt hermore, the results showed that enhancers obviously upregulate the expression of adjacent target genes which were remarkably correlated with the functions of corresponding tissues. Finally, a free user-friendly tissue-specific enhancer database, TiED (http://lcbb.swjtu.edu.cn/TiED), has been built to store, visualize, and confer these results. Conclusion: Genome-wide analysis of the regulatory network and characteristic of various types of enhancers showed that enhancers associated with TFs, eRNAs and target genes appeared in tissue specificity and function across different tissues.

Charge designable and tunable GFP as a target pH-responsive carrier for intracellular functional protein delivery and tracing.

A charge designable and tunable green fluorescent protein (GFP)-based protein delivery strategy was proposed. The acquired His29GFP selectively permeates the cell membrane at a target pH of 6.5 and escapes from the endosome efficiently. The delivered RNase A caused substantial mRNA degradation in HeLa cells, and proliferation inhibition in different cell lines and a 3D tumor model at pH 6.5.

The RhoA/ROCK pathway mediates high glucose-induced cardiomyocyte apoptosis via oxidative stress, JNK, and p38MAPK pathways.

AIMS: To understand the roles of the RhoA/ROCK and mitogen-activated protein kinase (MAPK) pathways in high glucose (HG)-induced apoptosis and oxidative stress in cardiomyocytes. MATERIALS AND METHODS: Neonatal rat cardiomyocytes were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 or 30 mmol/L D-glucose, in the presence or absence of fasudil (50 or 100 µM), SB203580, SP600125, or PD98059 (10 µM, respectively). The percentage of early apoptotic cardiomyocytes was evaluated using flow cytometry. The superoxide dismutase activity and malondialdehyde contents in the cellular supernatants were measured. The Bax and Bcl-2 mRNA levels were determined by quantitative real-time PCR. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1), p38MAPK, JNK, and ERK as well as the protein levels of Bax, Bcl-2, and cleaved caspase-3 was analysed by Western blot. RESULTS: Fasudil, SB203580, and SP600125 effectively inhibited the HG-induced early apoptosis increase and decreased Bax mRNA expression, the Bax/Bcl-2 protein expression ratio, and cleaved caspase-3 protein levels in the cardiomyocytes; this was accompanied by upregulation of the Bcl-2 mRNA. Moreover, fasudil markedly increased the superoxide dismutase activity level and suppressed the elevation in HG-induced malondialdehyde content and the phosphorylation of MYPT1, p38MAPK and JNK. CONCLUSIONS: The RhoA/ROCK pathway mediates HG-induced cardiomyocyte apoptosis via oxidative stress and activation of p38MAPK and JNK in neonatal rats in vitro. Fasudil effectively ameliorates HG-induced cardiomyocyte apoptosis by suppressing oxidative stress and the p38MAPK and JNK pathways.

Agmatine Reduces Lipopolysaccharide-Mediated Oxidant Response via Activating PI3K/Akt Pathway and Up-Regulating Nrf2 and HO-1 Expression in Macrophages.

Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation.

The small-molecule inhibitor selectivity between IKKα and IKKß kinases in NF-κB signaling pathway.

The enzyme complex IκB kinase (IKK) is an essential activator of NF-κB signaling pathway involved in propagating the cellular response to inflammation. The complex contains two functional subunits IKKα and IKKß, which are structurally conserved kinases and selective inhibition of them would result in distinct biological effects. However, most existing IKK inhibitors show moderate or high promiscuity for the two homologous kinases. Understanding of the molecular mechanism and biological implication underlying the specific interactions in IKK-ligand recognition is thus fundamentally important for the rational design of selective IKK inhibitors. In the current work, we integrated molecular docking, quantum mechanics/molecular mechanics calculation and Poisson-Boltzmann/surface area analysis to investigate the structural basis and energetic property of the selective binding of small-molecule ligands to IKKα and IKKß. It was found that the selectivity is primarily determined by the size and topology difference in ATP-binding pocket of IKKα and IKKß kinase domains; bulky inhibitor molecules commonly have, respectively, low and appropriate affinities towards IKKα and IKKß, and thus exhibit relatively high selectivity for IKKß over IKKα, whereas small ligands can only bind weakly to both the two kinases with low selectivity. In addition, the conformation, arrangement and distribution of residues in IKK pockets are also responsible for constituting the exquisite specificity of ligand binding to KKα and IKKß. Next, a novel quantitative structure-selectivity relationship model was developed to characterize the relative contribution of each kinase residue to inhibitor selectivity and to predict the selectivity and specificity for a number of known IKK inhibitors. Results showed that the active-site residues contribute significantly to the selectivity by directly interacting with inhibitor ligands, while those protein portions far away from the kinase active sites may also play an important role in determining the selectivity through long-range non-bonded forces and indirect allosteric effect.

Molecular docking characterization of a four-domain segment of human fibronectin encompassing the RGD loop with hydroxyapatite.

Fibronectin adsorption on biomaterial surfaces plays a key role in the biocompatibility of biomedical implants. In the current study, the adsorption behavior of the 7-10th type III modules of fibronectin (FN-III7-10) in the presence of hydroxyapatite (HAP) was systematically investigated by using molecular docking approach. It was revealed that the FN-III10 is the most important module among FN-III7-10 in promoting fibronectin binding to HAP by optimizing the interaction energy; the arginine residues were observed to directly interact with the hydroxyl group of HAP through electrostatic forces and hydrogen bonding. Moreover, it was found that the HAP-binding sites on FN-III10 are mainly located at the RGD loop region, which does not affect the interaction between the fibronectin protein and its cognate receptors on the cell surface.