An intelligent DNA nanomachine for amplified MicroRNA imaging and MicroRNA-Guided efficient gene silencing.

The DNA nanomachines as excellent synthetic biological tools have been widely used for the sensitive detection of intracellular microRNA (miRNA) and DNAzyme-involved gene silencing. However, intelligent DNA nanomachines which have the ability to sense intracellular specific biomolecules and respond to external information in complex environments still remain challenging. Herein, we develop a miRNA-responsive DNAzyme cascaded catalytic (MDCC) nanomachine to perform multilayer cascade reactions, enabling the amplified intracellular miRNA imaging and miRNA-guided efficient gene silencing. The intelligent MDCC nanomachine is designed based on multiple DNAzyme subunit-encoded catalyzed hairpin assembly (CHA) reactants sustained by the pH-responsive Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles. After cellular uptake, the MDCC nanomachine degrades in acidic endosome and releases three hairpin DNA reactants and Zn2+, and the latter can act as an effective cofactor for DNAzyme. In the presence of miRNA-21, a catalytic hairpin assembly (CHA) reaction is triggered, which produces a large number of Y-shaped fluorescent DNA constructs containing three DNAzyme modules for gene silencing. The construction of Y-shaped DNA modified with multisite fluorescence and the circular reaction realizes ultrasensitive miRNA-21 imaging of cancer cells. Moreover, miRNA-guided gene silencing inhibits the cancer cell proliferation through the DNAzyme-specific recognition and cleavage of target EGR-1 (Early Growth Response-1) mRNA, which is one key tumor-involved mRNA. The strategy may provide a promising platform for highly sensitive determination of biomolecules and accurate gene therapy of cancer cells.

Combined with UPLC-Triple-TOF/MS-based plasma lipidomics and molecular pharmacology reveals the mechanisms of schisandrin against Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD), a type of neurodegeneration disease, is characterized by Aß deposition and tangles of nerve fibers. Schisandrin is one of the main components of Fructus Schisandrae Chinensis. Researches showed that schisandrin can improve the cognitive impairment and memory of AD mice, but the specific mechanism has not been fully elucidated. PURPOSE: The purpose of this study is to investigate the possible mechanism of schisandrin in improving AD pathology. METHODS: The Morris water maze test was executed to detect spatial learning and memory. Ultra performance liquid chromatography-Triple time of flight mass spectrometry (UPLC-Triple-TOF/MS)-based plasma lipidomics was used to study the changes of plasma lipids. Moreover, we measured the levels of protein and mRNA expression of APOE and ABCA1 in the rat brains and in BV2 microglia. RESULTS: Our study found that schisandrin could improve learning and memory, and reduce Aß deposition in AD rats. Furthermore, we found that schisandrin can improve plasma lipid metabolism disorders. Therefore, we hypothesized schisandrin might act via LXR and the docking results showed that schisandrin interacts with LXRß. Further, we found schisandrin increased the protein and mRNA expression of LXR target genes APOE and ABCA1 in the brain of AD rats and in BV2 microglia. CONCLUSION: Our study reveals the neuroprotective effect and mechanism of schisandrin improves AD pathology by activating LXR to produce APOE and ABCA1.

Intelligent Programmable DNA Nanomachines for the Spatially Controllable Imaging of Intracellular MicroRNA.

The high programmability of DNA molecules makes them particularly suitable for constructing artificial molecular machines to perform sophisticated functions by simulating complex living systems. However, intelligent DNA nanomachines which can perform precise tasks logically in complex environments still remain challenging. Herein, we develop a general strategy to design a pH-responsive programmable DNA (PRPD) nanomachine to perform multilayer DNA cascades, enabling precise sensing and calculation of intracellular biomolecules. The PRPD nanomachine is built on a four-stranded DNAzyme walker precursor with a DNA switch on the surface of an Au nanoparticle, which is capable of precisely responding to pH variations in living cells by sequence tuning. This multilayer DNA cascade networks have been applicated in spatially controlled imaging of intracellular microRNA, which efficiently avoided the DNA nanomachine activated by nonspecific extracellular molecules and achieved apparent signal amplification. Our strategy enables the sensing-computing-output functional integration of DNA nanomachines, facilitating the application of programmable and complex nanomachines in nanoengineering, chemistry, and biomedicine.

Living-Cell MicroRNA Imaging with Self-Assembling Fragments of Fluorescent Protein-Mimic RNA Aptamer.

As the cellular roles of RNA abundance continue to increase, there is an urgent need for the corresponding tools to elucidate native RNA functions and dynamics, especially those of short, low-abundance RNAs in live cells. Fluorescent RNA aptamers provide a useful strategy to create the RNA tag and biosensor devices. Corn, which binds with 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), is a good candidate for the RNA tag because of its enhanced photostability and red-shifted spectrum. Herein, we report for the first time the utilization of Corn as a split aptamer system, combined with RNA-initiated fluorescence complementation (RIFC), for monitoring RNA self-assembly and sensing microRNA. In this platform, the 28-nt Corn was divided into two nonfunctional halves (named probe I and probe II), and an additional target RNA recognition and stem part was introduced in each probe. The target RNA can trigger the self-assembly reconstitution of the Corn's G-quadruplex scaffold for DFHO binding and turn-on fluorescence. These probes can be transfected stably into mammalian cells and deliver the light-up fluorescent response to microRNA-21 (miR-21). Significantly, the probes have good photostability, with minimal fluorescence loss after continuous irradiation, and can be used for imaging of miR-21 in living mammalian cells. The proposed method is universal and could be applied to the sensing of other tumor-associated RNAs, including messenger RNA and noncoding RNA, as well as for monitoring RNA/RNA interactions. The Corn-based splitting aptamers show promising potential in the real-time visualization and mechanistic analysis of nucleic acids.

Simultaneous monitoring of action potentials and neurotransmitter release from neuron-like PC12 cells.

Simultaneous recording of action potentials (APs) and neurotransmitter release is highly desirable in living neurons since it provides a complete framework of the physiological and pathological statuses of nerve cells. In this work, we proposed an approach coupling ultra-thin microelectrode array (MEA) with total internal reflection fluorescence microscopy (TIRFM), which served as a powerful platform to visualize both APs and vesicular exocytosis in a neuronal circuit model formed by neuron-like PC12 cells. Taking advantages of fluorescent false neurotransmitter (FFN), the transient neurotransmitter transport down an axon could be visualized with high spatial and temporal resolution. The real-time recording of APs burst and neurotransmitter release induced by hypoxia with MEA/TIRFM platform reveals the relevance of electrical and chemical activities in the neuronal model. The combination of the optical and electrical techniques enables mapping of neuron connectivity in an entire neuronal circuit, which may ultimately lead to deeper understanding of nervous system.

Acid-Switchable DNAzyme Nanodevice for Imaging Multiple Metal Ions in Living Cells.

Metal-assisted deoxyribozyme catalysis (DNAzyme) has been a general platform for constructing highly sensitive and selective detection sensors of metal ions. However, the "always on" mode of the traditional DNAzyme sensors greatly limits their application in the visual analysis of endogenous metal ions in a complex physiological microenvironment. To overcome this obstacle, a smart acid-switchable DNAzyme nanodevice is designed to control the DNAzyme activity in living cells and achieve simultaneous visualization of metal ions (Zn2+ and Pb2+) in situ. This nanodevice is built on DNAzyme precursors (DPs) and acid-switchable DNA (SW-DNA), precisely responding to pH variations in the range of 4.5-7.0, and the state of the three-strand hybridization of DPs successfully renders the DNAzymes inactive before being transported into cells. Once the nanodevice is taken up into living cells, the SW-DNA will change the configuration from linear to triplex in the acidic intracellular compartments (lysosomes, pH ∼4.5 to 5.0) and then the strands hybridized with the SW-DNA are liberated and subsequently react with DPs to form the active DNAzyme, which can further realize multi-imaging of intracellular metal ions. Moreover, this strategy has broad prospects as a powerful platform for constructing various acid-switchable nanodevices for visual analysis of multiple biomolecules in living cells.

NIR Remote-Controlled "Lock-Unlock" Nanosystem for Imaging Potassium Ions in Living Cells.

Despite great achievements in sensitive and selective detection of important biomolecules in living cells, it is still challenging to develop smart and controllable sensing nanodevices for cellular studies that can be activated at desired time in target sites. To address this issue, we have constructed a remote-controlled "lock-unlock" nanosystem for visual analysis of endogenous potassium ions (K+), which employed a dual-stranded aptamer precursor (DSAP) as recognition molecules, SiO2 based gold nanoshells (AuNS) as nanocarriers, and near-infrared ray (NIR) as the remotely applied stimulus. With the well-designed and activatable DSAP-AuNS, the deficiencies of traditional aptamer-based sensors have been successfully overcome, and the undesired response during transport has been avoided, especially in complex physiological microenvironments. While triggered by NIR, the increased local temperature of AuNS induced the dehybridiztion of DSAP, realized the "lock-unlock" switch of the DSAP-AuNS nanosystem, activated the binding capability of aptamer, and then monitored intracellular K+ via the change of fluorescence signal. This DSAP-AuNS nanosystem not only allows us to visualize endogenous ions in living cells at a desired time but also paves the way for fabricating temporal controllable nanodevices for cellular studies.

A self-powered 3D DNA walker with programmability and signal-amplification for illuminating microRNA in living cells.

Based on the structural programmability and spatial addressability of DNA nanodevices, a target-triggered, enzyme-free 3D DNA walker, comprising of hairpin DNA assembled gold nanoparticles with a local catalytic hairpin assembly reaction, was developed for the highly sensitive detection of intracellular tumor-associated microRNAs.

Target-triggered, self-powered DNAzyme-MnO2 nanosystem: towards imaging microRNAs in living cells.

A target-triggered, self-powered strategy for in situ monitoring of intracellular microRNAs was fabricated via a versatile DNAzyme-MnO2 nanosystem, which integrates delivery, quenching, target recognition, self-supply of cofactors and signal amplification all in one.