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.

CRIP1 involves the pathogenesis of multiple myeloma via dual-regulation of proteasome and autophagy.

BACKGROUND: Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment. METHODS: The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells. FINDINGS: High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. In vitro and in vivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200. INTERPRETATION: Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM. FUNDING: A full list of funding sources can be found in the acknowledgements section.

Striking a growth-defense balance: Stress regulators that function in maize development.

Maize (Zea mays) cultivation is strongly affected by both abiotic and biotic stress, leading to reduced growth and productivity. It has recently become clear that regulators of plant stress responses, including the phytohormones abscisic acid (ABA), ethylene (ET), and jasmonic acid (JA), together with reactive oxygen species (ROS), shape plant growth and development. Beyond their well established functions in stress responses, these molecules play crucial roles in balancing growth and defense, which must be finely tuned to achieve high yields in crops while maintaining some level of defense. In this review, we provide an in-depth analysis of recent research on the developmental functions of stress regulators, focusing specifically on maize. By unraveling the contributions of these regulators to maize development, we present new avenues for enhancing maize cultivation and growth while highlighting the potential risks associated with manipulating stress regulators to enhance grain yields in the face of environmental challenges.

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.

Phosphorescent-Dye-Sensitized Quantum-Dot Light-Emitting Diodes with 37% External Quantum Efficiency.

Exciton harvesting is of paramount importance for quantum-dot light-emitting diodes (QLEDs). Direct exciton harvesting by the quantum dots (QDs) emitting layer suffers from poor hole injection due to the low conduction bands and valence bands of QDs, leading to unbalanced electron-hole injection and recombination. To address this issue, here, an exciton sensitizing approach is reported, where excitons form on a phosphorescent-dye-doped layer, which then transfer their energies to adjacent QDs layer for photon emission. Due to the very efficient exciton formation and energy-transfer processes, higher device performance can be achieved. To demonstrate the above strategy, red QLEDs with a phosphorescent dye, iridium (III) bis(2-methyldibenzo-[f,h]quinoxaline) (acetylacetonate), Ir(MDQ)2 (acac), doped hole-transporting layer are fabricated and studied. At a doping concentration of 10 wt%, the best device achieves record high current efficiency, power efficiency, and external quantum efficiency (EQE) of 37.3 cd A-1 , 41 lm W-1 , and 37%, respectively. Simultaneously, the efficiency roll-off characteristic is greatly improved, in that 35% EQE can be well retained at a high luminance level of 450 000 cd m-2 . Moreover, the devices also exhibit good stability and reproducibility.

Exosome miRNAs profiling in serum and prognostic evaluation in patients with multiple myeloma.

MicroRNAs (MiRNAs) carried by exosomes play pivotal roles in the crosstalk between cell components in the tumor microenvironment. Our study aimed at identifying the expression profile of exosomal miRNAs (exo-miRNAs) in the serum of multiple myeloma (MM) patients and investigating the regulation networks and their potential functions by integrated bioinformatics analysis. Exosomes in serum from 19 newly diagnosed MM patients and 9 healthy donors were isolated and the miRNA profile was investigated by small RNA sequencing. Differential expression of exo-miRNAs was calculated and target genes of miRNAs were predicted. CytoHubba was applied to identify the hub miRNAs and core target genes. The LASSO Cox regression model was used to develop the prognostic model, and the ESTIMATE immune score was calculated to investigate the correlation between the model and immune status in MM patients. The top six hub differentially expressed serum exo-miRNAs were identified. 513 target genes of the six hub exo-miRNAs were confirmed to be differentially expressed in MM cells in the Zhan Myeloma microarray dataset. Functional enrichment analysis indicated that these target genes were mainly involved in mRNA splicing, cellular response to stress, and deubiquitination. 13 core exo-miRNA target genes were applied to create a novel prognostic signature to provide risk stratification for MM patients, which is associated with the immune microenvironment of MM patients. Our study comprehensively investigated the exo-miRNA profiles in MM patients. A novel prognostic signature was constructed to facilitate the risk stratification of MM patients with distinct outcomes.

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.

Husband-wife Relationship, Neonatal Health, Breast Milk Volume and Postpartum Depression: A Prospective Cohort Study.

Postpartum depression (PPD) is a major public health problem that has negative effects on mothers, infants, and society. This study was aimed at investigating the prevalence of PPD and elucidating the delivery factors implicated in PPD so as take more targeted measures for reducing the potential risk factors. A prospective cohort study was conducted. Following the criterion, 151 pregnant women were included in the study. The Edinburgh Postpartum Depression Scale (EPDS) and the general questionnaire were filled out 2-3 days after delivery. At weeks 2 and 6 postpartum, the EPDS was reassessed either online or via telephone. Also, electronic medical records based on relevant information during the delivery period were collected. Statistical significance was defined as p < 0.05. A high rate of PPD (31.13%) was reported. Univariate correlation analysis showed statistically significant differences in the husband-wife relationship (χ2 = 18.497, p < 0.001), neonatal health (χ2 = 14.710, p < 0.001), and breast milk volume (χ2 = 5.712, p = 0.017) between PPD and normal control groups. Adjusting for other covariates, multivariate logistic regression analysis showed that satisfactory conjugal relation could reduce the risk of PPD (OR, 0.053; p = 0.022); Neonatal health problems significantly increase the risk of PPD (OR, 6.497; p = 0.001); Adequate breast milk could alleviate the risk of PPD (OR, 0.351; P = 0.045). Data analysis suggests that marital discord and unhealthy new-born are independent risk factors; nevertheless, sufficient breast milk is a protective factor against PPD. Healthcare workers such as hospital and community doctors and social workers should pay attention to PPD. Furthermore, perinatal emotional support, health education, and EPDS assessment need to be incorporated into maternity care. Screening and personalized psychological counselling should be carried out for high-risk pregnant women with PPD.

Maize LOST SUBSIDIARY CELL encoding a large subunit of ribonucleotide reductase is required for subsidiary cell development and plant growth.

The four-celled stomatal complex consists of a pair of guard cells (GCs) and two subsidiary cells (SCs) in grasses, which supports a fast adjustment of stomatal aperture. The formation and development of SCs are thus important for stomatal functionality. Here, we report a maize lost subsidiary cells (lsc) mutant, with many stomata lacking one or two SCs. The loss of SCs is supposed to have resulted from impeded subsidiary mother cell (SMC) polarization and asymmetrical division. Besides the defect in SCs, the lsc mutant also displays a dwarf morphology and pale and striped newly-grown leaves. LSC encodes a large subunit of ribonucleotide reductase (RNR), an enzyme involved in deoxyribonucleotides (dNTPs) synthesis. Consistently, the concentration of dNTPs and expression of genes involved in DNA replication, cell cycle progression, and SC development were significantly reduced in the lsc mutant compared with the wild-type B73 inbred line. Conversely, overexpression of maize LSC increased dNTP synthesis and promoted plant growth in both maize and Arabidopsis. Our data indicate that LSC regulates dNTP production and is required for SMC polarization, SC differentiation, and growth of maize.

Resveratrol alleviates postpartum depression-like behavior by activating autophagy via SIRT1 and inhibiting AKT/mTOR pathway.

BACKGROUND: Postpartum depression (PPD) causes maternal mortality, and has a high disability rate. In recent years, studies have suggested the Sirt1 gene to be involved in the pathogenesis of depression. Resveratrol (RSV), an activator of Sirt1, has been investigated in depressive behavior. However, its effect on PPD remains to be thoroughly elucidated. METHODS: We employed a mice model with bilateral oophorectomy combined with hormone-simulated pregnancy to assess postpartum depression-like behavior. The behavioral tests were performed 2 days after the withdrawal of estradiol benzoate. RSV was administered subcutaneously to the PPD model mice. Several behavioral tests were executed, including the open field test, forced swimming test, and tail suspension test. Western blot analyses and immunofluorescence staining were used to evaluate protein expression levels of SIRT1, autophagy markers, and the AKT/mTOR. RESULTS: Postpartum depressive-like behavior was triggered following the withdrawal of estradiol benzoate after hormone-stimulated-pregnancy. RSV improved postpartum depressive-like behavior of mice via its upregulation of the SIRT1 and autophagy markers, such as Beclin1, ATG5 and LC3B. Also, the downregulation of the p62 protein expression was observed. More importantly, we also detected the inhibition of phosphorylated AKT and mTOR in the hippocampus of postpartum depressive-like mice. CONCLUSION: RSV could alleviate postpartum depression-like behavior in mice by stimulating the SIRT1, induce autophagy and inhibit the AKT/ mTOR signaling pathway.

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.

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.

The association of oxytocin with major depressive disorder: role of confounding effects of antidepressants.

Major depressive disorder is a genetic susceptible disease, and a psychiatric syndrome with a high rate of incidence and recurrence. Because of its complexity concerning etiology and pathogenesis, the cure rate of first-line antidepressants is low. In recent years, accumulative evidences revealed that oxytocin act as a physiological or pathological participant in a variety of complex neuropsychological activities, including major depressive disorder. Six electronic databases (Web of Science, PubMed, Scopus, Google Scholar, CNKI, and Wanfang) were employed for researching relevant publications. At last, 226 articles were extracted. The current review addresses the correlation of the oxytocin system and major depressive disorder. Besides, we summarize the mechanisms by which the oxytocin system exerts potential antidepressant effects, including regulating neuronal activity, influencing neuroplasticity and regeneration, altering neurotransmitter release, down regulating hypothalamic-pituitary-adrenal axis, anti-inflammatory, antioxidation, and genetic effects. Increasing evidence shows that oxytocin and its receptor gene may play a potential role in major depressive disorder. Future research should focus on the predictive ability of the oxytocin system as a biomarker, as well as its role in targeted prevention and early intervention of major depressive disorder.

Amplified and label-free electrochemical detection of a protease biomarker by integrating proteolysis-triggered transcription.

Cell-free synthetic biology provides a promising strategy for developing high-performance biosensors by integrating with advanced testing technologies. However, the combination of synthetic biology with electrochemical testing techniques is still underdeveloped. Here, we proposed an electrochemical biosensor for the label-free and ultrasensitive detection of target protease biomarker by coupling a protease-responsive RNA polymerase (PR) for signal amplification. Taking tumor biomarker matrix metalloprotease-2 (MMP-2) as a model protease, we employed PR to transduce each proteolysis reaction mediated by MMP-2 into multiple programmable RNA outputs that can be captured by the DNA probes immobilized on a gold electrode. Moreover, the captured RNAs are designed to contain a guanine-rich sequence that can form G-quadruplex and bind to hemin in the presence of potassium ions. In this scenario, the activity of MMP-2 is converted and amplified into the electrochemical signals of hemin. Under the optimal conditions, this PR-based electrochemical biosensor enabled the sensitive detection of MMP-2 in a wide linear dynamic range from 10 fM to 1.0 nM, with a limit of detection of 7.1 fM. Moreover, the proposed biosensor was further applied in evaluating MMP-2 activities in different cell cultures and human tissue samples, demonstrating its potential in the analysis of protease biomarkers in complex clinical samples.

A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics.

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas-powered catalytic nucleic acid circuit, namely, CRISPR-Cas-only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer-associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

A Missense Mutation in a Large Subunit of Ribonucleotide Reductase Confers Temperature-Gated Tassel Formation.

Temperature is a major factor regulating plant growth. To reproduce at extreme temperatures, plants must develop normal reproductive organs when exposed to temperature changes. However, little is known about the underlying molecular mechanisms. Here, we identified the maize (Zea mays) mutant thermosensitive vanishing tassel1-R (tvt1-R), which lacks tassels at high (restrictive) temperatures due to shoot apical meristem (SAM) arrest, but forms normal tassels at moderate (permissive) temperatures. The critical stage for phenotypic conversion in tvt1-R mutants is V2 to V6 (Vn, where "n" is the number of leaves with collars visible). Positional cloning and allelism and complementation tests revealed that a G-to-A mutation causing a Arg277-to-His277 substitution in ZmRNRL1, a ribonucleotide reductase (RNR) large subunit (RNRL), confers the tvt1-R mutant phenotype. RNR regulates the rate of deoxyribonucleoside triphosphate (dNTP) production for DNA replication and damage repair. By expression, yeast two-hybrid, RNA sequencing, and flow cytometric analyses, we found that ZmRNRL1-tvt1-R failed to interact with all three RNR small subunits at 34°C due to the Arg277-to-His277 substitution, which could impede RNR holoenzyme (α2ß2) formation, thereby decreasing the dNTP supply for DNA replication. Decreased dNTP supply may be especially severe for the SAM that requires a continuous, sufficient dNTP supply for rapid division, as demonstrated by the SAM arrest and tassel absence in tvt1-R mutants at restrictive temperatures. Our study reveals a novel mechanism of temperature-gated tassel formation in maize and provides insight into the role of RNRL in SAM maintenance.

Optimized integration of fluoxetine and 7, 8-dihydroxyflavone as an efficient therapy for reversing depressive-like behavior in mice during the perimenopausal period.

Fluoxetine (FLX) has been considered as an effective anti-depressant drug. Besides, previous studies reported reasonable anti-depressant effects for 7, 8-dihydroxyflavone (7, 8 DHF). However, the combination of FLX and 7, 8 DHF in a well-established depression model has not been explored. In this study, we demonstrate that the 7, 8 DHF can improve the anti-depressant efficacy of FLX in a chronic unpredictable mild stress (CUMS)-induced depression during the perimenopausal period. The corresponding mechanism of FLX+7, 8 DHF therapy and the effect of ANA-12 are also investigated. Moreover, the influences of 7, 8 DHF (5 mg/kg/day), FLX (18 mg/kg/day), and ANA-12 (0.5 mg/kg/day) on a depressive-like behavior are displayed. Inflammatory, autophagic and apoptotic changes of hippocampus and cortex are examined by using western blot, immunofluorescence, and Real-Time Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) techniques. The protein levels of phosphatidylinositol 3 kinase (PI3K)/ protein kinase B (Akt)/mechanistic target of rapamycin (mTOR)/phosphorylated extracellular signal-regulated kinase1/2 (p-ErK 1/2)/brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) of hippocampus and cortex are assessed by western blot. The combined FLX and 7, 8 DHF treatment can significantly improve depressive-like behavior in sucrose preference and forced swimming tests accompanied by a noticeable upregulation of autophagy, neuronal nuclei (NeuN), ionized calcium-binding adaptor molecule 1 (Iba1) expressions, and PI3K/Akt/ mTOR/ p-ErK 1/2 signaling pathways. Besides, an obvious increase of the brain-derived neurotrophic factor (BDNF) and TrkB levels are observed with down-regulated inflammation and apoptosis. These findings suggest that the integrated FLX and 7, 8 DHF holds a potential as an efficient treatment to ameliorate depressive-like behavior in perimenopausal patients.

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.

Engineering Cell-Surface Receptors with DNA Nanotechnology for Cell Manipulation.

Cell-surface receptors play pivotal roles in the regulation of cell fate. Molecular engineering of cell-surface receptors enables control of cell signaling and manipulation of cell behavior in a user-defined way. Currently, the development of chemical-biological approaches for non-genetic engineering and regulation of membrane receptors is attracting significant interest. Recent research advances in functional nucleic acids and DNA nanotechnology have made it possible to use DNA as a new and promising molecular toolkit for controlling receptor-mediated signaling and cell fates. In this minireview we summarize the advances in the use of DNA nanotechnology for the spatiotemporal regulation of cell receptors and highlight practical applications in manipulating cell functions including cell adhesion, cell-cell contact, cell migration, and cellular immunity. We also provide a perspective on the potential of and challenges facing DNA-based receptor engineering in future applications of cell manipulation and cell-based therapy.