In Situ Spatial Complementation of Aptamer‐Mediated Recognition Enables Live‐Cell Imaging of Native RNA Transcripts in Real Time

Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single‐cell resolution. In contrast to routine fluorescent‐protein‐based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology. Herein, we report the development of an aptamer‐initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)‐mimicking turn‐on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, endogenous mRNA molecules recruited Split‐Broccoli and brought the two fragments into spatial proximity, which formed a fluorophore‐binding site in situ and turned on fluorescence. Significantly, we demonstrated the use of AiFC for high‐contrast and real‐time imaging of endogenous RNA molecules in living mammalian cells. We envision wide application and practical utility of this enabling technology to in vivo single‐cell visualization and mechanistic analysis of macromolecular interactions.     

Fluorophore‐Promoted RNA Folding and Photostability Enables Imaging of Single Broccoli‐Tagged mRNAs in Live Mammalian Cells

Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli‐DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli‐BI complexes in cells. Broccoli‐BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis‐BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.     

Muropeptides in Pseudomonas aeruginosa and their Role as Elicitors of β‐Lactam‐Antibiotic Resistance

Muropeptides are a group of bacterial natural products generated from the cell wall in the course of its turnover. These compounds are cell‐wall recycling intermediates and are also involved in signaling within the bacterium. However, the identity of these signaling molecules remains elusive. The identification and characterization of 20 muropeptides from Pseudomonas aeruginosa is described. The least abundant of these metabolites is present at 100 and the most abundant at 55,000 molecules per bacterium. Analysis of these muropeptides under conditions of induction of resistance to a β‐lactam antibiotic identified two signaling muropeptides (N‐acetylglucosamine‐1,6‐anhydro‐N‐acetylmuramyl pentapeptide and 1,6‐anhydro‐N‐acetylmuramyl pentapeptide). Authentic synthetic samples of these metabolites were shown to activate expression of β‐lactamase in the absence of any β‐lactam antibiotic, thus indicating that they serve as chemical signals in this complex biochemical pathway.     

Patterned Paper Sensors Printed with Long‐Chain DNA Aptamers

There is growing interest in developing printable paper sensors to enable rapid testing of analytes for environmental, food safety, and clinical applications. A major challenge is to find suitable bioinks that are amenable to high‐speed printing and remain functional after printing. We report on a simple and effective approach wherein an aqueous ink composed of megadalton‐sized tandem repeating structure‐switching DNA aptamers (concatemeric aptamers) is used to rapidly create patterned paper sensors on filter paper by inkjet printing. These concatemeric aptamer reporters remain immobilized at the point of printing through strong adsorption but retain sufficient segmental mobility to undergo structure switching and fluorescence signaling to provide both qualitative and quantitative detection of small molecules and protein targets. The convenience of inkjet printing allows for the patterning of internally referenced sensors with multiplexed detection, and provides a generic platform for on‐demand printing of sensors even in remote locations.     

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.     

High‐Throughput Isolation of Cell Protrusions with Single‐Cell Precision for Profiling Subcellular Gene Expression

Invading cancer cells extend cell protrusions, which guide cancer‐cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single‐cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high‐throughput isolation of cell protrusions at single‐cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell‐protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single‐cell precision for subsequent RNA‐Seq analysis, which was further validated by RT‐qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.     

MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures

One major challenge in nucleic acids analysis by hybridization probes is a compromise between the probe's tight binding and sequence‐selective recognition of nucleic acid targets folded into stable secondary structures. We have been developing a four‐way junction (4WJ)‐based sensor that consists of a universal stem‐loop (USL) probe immobilized on an electrode surface and two adaptor strands (M and F). The sensor was shown to be highly selective towards single base mismatches at room temperature, able to detect multiple targets using the same USL probe, and have improved ability to detect folded nucleic acids. However, some nucleic acid targets, including natural RNA, are folded into very stable secondary and tertiary structures, which may represent a challenge even for the 4WJ sensors. This work describes a new sensor, named MVF since it uses three probe stands M, V and F, which further improves the performance of 4WJ sensors with folded targets. The MVF sensor interrogating a 16S rRNA NASBA amplicon with calculated folding energy of ?32.82 kcal/mol has demonstrated 2.5‐fold improvement in a signal‐to‐background ratio in comparison with a 4WJ sensor lacking strand V. The proposed design can be used as a general strategy in the analysis of folded nucleic acids including natural RNA.     

Highly Multiplexed Single‐Cell In Situ Protein Analysis with Cleavable Fluorescent Antibodies

Limitations on the number of proteins that can be quantified in single cells in situ impede advances in our deep understanding of normal cell physiology and disease pathogenesis. Herein, we present a highly multiplexed single‐cell in situ protein analysis approach that is based on chemically cleavable fluorescent antibodies. In this method, antibodies tethered to fluorophores through a novel azide‐based cleavable linker are utilized to detect their protein targets. After fluorescence imaging and data storage, the fluorophores coupled to the antibodies are efficiently cleaved without loss of protein target antigenicity. Upon continuous cycles of target recognition, fluorescence imaging, and fluorophore cleavage, this approach has the potential to quantify over 100 different proteins in individual cells at optical resolution. This single‐cell in situ protein profiling technology will have wide applications in signaling network analysis, molecular diagnosis, and cellular targeted therapies.     

Electrochemical Sensor Prepared from Molecularly Imprinted Polymer for Recognition of 1,3-Dinitrobenzene (DNB)

An electrochemical sensor was modified with multi‐wall carbon nanotubes (MWCNT) and molecularly imprinted polymer (MIP) material synthesized with acrylamide and ethylene glycol dimethacrylate (EGDMA) in the presence of 1,3‐dinitrobenzene (DNB) as the template molecule. The MWCNT and MIP layers were successively modified on the surface of a glassy carbon electrode (GCE), of which the MIP film works as an artificial receptor due to its specific molecular recognition sites. The MIP material was characterized by FT‐IR and electrochemical methods of square wave voltammetry (SWV). The interferences of other nitroaromatic compounds (NAC) such as 2,4,6‐trinitrotoluene (TNT), 1,3,5‐trinitrobenzene (TNB) and 2,4‐dinitrotoluene (DNT) to DNB were also investigated by the prepared MIP/MWCNT electrode. Compared with other traditional sensors, the MIP/MWCNT modified electrode shows good selectivity and sensitivity. In addition, the current responses to DNB are linear with the concentration ranging from 4.5×10^?8 to 8.5×10^?6 mol/L with the detection limits of 2.5×10^?8 (?0.58 V) and 1.5×10^?8 mol/L (?0.69 V) (S/N=3). The construction process of MIP/MWCNT modified electrode was also studied as well. All results indicate that the MIP/MWCNT modified electrode established an improving way for simple, fast and selective analysis of DNB.     

Intrinsic Electrocatalysis of RNA as a Label‐free and Reagent‐less Tool for Detection of MicroRNAs

We show for the first time that RNA catalyzes hydrogen evolution reaction at mercury‐containing electrodes. We previously showed that DNA is electrocatalytically active, and so we compared heights and potentials of the RNA chronopotentimetric stripping (CPS) peaks with analogous signals of DNA of the same sequence and found out they were very similar. RNA peaks showed differences depending on the RNA base composition. Catalytic nature of CPS peak enabled detection of 25 pM microRNA in electrochemical cell, or 500 pM microRNA in a 5 μL solution drop (corresponding to 2.5 fmole of microRNA). This finding opens the door for simple, label‐free and reagent‐less analysis of low concentrations of RNA molecules.     

A Stretchable Electrochemical Sensor for Inducing and Monitoring Cell Mechanotransduction in Real Time

Existing methods offer little direct and real‐time information about stretch‐triggered biochemical responses during cell mechanotransduction. A novel stretchable electrochemical sensor is reported that takes advantage of a hierarchical percolation network of carbon nanotubes and gold nanotubes (CNT‐AuNT). This hybrid nanostructure provides the sensor with excellent time‐reproducible mechanical and electrochemical performances while granting very good cellular compatibility, making it perfectly apt to induce and monitor simultaneously transient biochemical signals. This is validated by monitoring stretch‐induced transient release of small signaling molecules by both endothelial and epithelial cells cultured on this sensor and submitted to stretching strains of different intensities. This work demonstrates that the hybrid CNT‐AuNT platform offers a versatile and highly sensitive way to characterize and quantify short‐time mechanotransduction responses.     

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor,Neratinib, in Cancer Cells

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label‐free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC‐MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.     

Simultaneous In Situ Quantification of Two Cellular Lipid Pools Using Orthogonal Fluorescent Sensors

Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid‐binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems‐level investigation of lipid‐mediated cell signaling and regulation.     

RNA aptamers as pathway-specific MAP kinase inhibitors

BACKGROUND: In eukaryotic cells, many intracellular signaling pathways have closely related mitogen activated protein kinase (MAPK) paralogs as central components. Although MAPKs are therefore obvious targets to control the cellular responses resulting from the activation of these signaling pathways, the development of inhibitors which target specific cell signaling pathways involving MAPKs has proven difficult. RESULTS: We used an RNA combinatorial approach to isolate RNAs that inhibit the in vitro phosphorylation activity of extracellular regulated kinase 2 (ERK2). These inhibitors block phosphorylation by ERK1 and ERK2, but do not inhibit Jun N-terminal kinase or p38 MAPKs. Kinetic analysis indicates these inhibitors function at high picomolar concentrations through the steric exclusion of substrate and ATP binding. In one case, we identified a compact RNA structural domain responsible for inhibition. CONCLUSIONS: RNA reagents can selectively recognize and inhibit MAPKs involved in a single signal transduction pathway. The methodology described here is readily generalizable, and can be used to develop inhibitors of MAPKs involved in other signal transduction pathways. Such reagents may be valuable tools to analyze and distinguish homologous effectors which regulate distinct signaling responses.     

Fluorophore-Promoted RNA Folding and Photostability Enables Imaging of Single Broccoli-Tagged mRNAs in Live Mammalian Cells

Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli-DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli-BI complexes in cells. Broccoli-BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis-BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.     

Capillary waveguide nucleic acid based biosensor

Micro-capillaries are finding increasing utility in the development of portable analytical sensors. We present design guidelines for optimizing the collection of free propagating fluorescence for capillary waveguide sensors used in the detection of nucleic acids. A dual function integrated opto/fluid connector is also described. Evanescent wave excitation of the coating layer containing a DNA probe is achieved by using a fiber optic ring arrangement for coupling light directly into the capillary wall. The central part of the connector is used for injecting a DNA or RNA target into the capillary channel. In situ hybridization has been used to detect target molecules at a concentration of 30 pg ml⁻¹. The sensor can be regenerated for repeated detection of DNA or RNA targets.     

Mobile trap algorithm for zinc detection using protein sensors

We present a mobile trap algorithm to sense zinc ions using protein-based sensors such as carbonic anhydrase (CA). Zinc is an essential biometal required for mammalian cellular functions although its intracellular concentration is reported to be very low. Protein-based sensors like CA molecules are employed to sense rare species like zinc ions. In this study, the zinc ions are mobile targets, which are sought by the mobile traps in the form of sensors. Particle motions are modeled using random walk along with the first passage technique for efficient simulations. The association reaction between sensors and ions is incorporated using a probability (p1) upon an ion-sensor collision. The dissociation reaction of an ion-bound CA molecule is modeled using a second, independent probability (p2). The results of the algorithm are verified against the traditional simulation techniques (e.g., Gillespie's algorithm). This study demonstrates that individual sensor molecules can be characterized using the probability pair (p1,p2), which, in turn, is linked to the system level chemical kinetic constants, kon and koff. Further investigations of CA-Zn reaction using the mobile trap algorithm show that when the diffusivity of zinc ions approaches that of sensor molecules, the reaction data obtained using the static trap assumption differ from the reaction data obtained using the mobile trap formulation. This study also reveals similar behavior when the sensor molecule has higher dissociation constant. In both the cases, the reaction data obtained using the static trap formulation reach equilibrium at a higher number of complex molecules (ion-bound sensor molecules) compared to the reaction data from the mobile trap formulation. With practical limitations on the number sensors that can be inserted/expressed in a cell and stochastic nature of the intracellular ionic concentrations, fluorescence from the number of complex sensor molecules at equilibrium will be the measure of the intracellular ion concentration. For reliable detection of zinc ions, it is desirable that the sensors must not bind all the zinc ions tightly, but should rather bind and unbind. Thus for a given fluorescence and with association-dissociation reactions between ions and sensors, the static trap approach will underestimate the number of zinc ions present in the system.     

Coronavirus Infection-Associated Cell Death Signaling and Potential Therapeutic Targets

COVID-19 is the name of the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that occurred in 2019. The virus–host-specific interactions, molecular targets on host cell deaths, and the involved signaling are crucial issues, which become potential targets for treatment. Spike protein, angiotensin-converting enzyme 2 (ACE2), cathepsin L-cysteine peptidase, transmembrane protease serine 2 (TMPRSS2), nonstructural protein 1 (Nsp1), open reading frame 7a (ORF7a), viral main protease (3C-like protease (3CLpro) or Mpro), RNA dependent RNA polymerase (RdRp) (Nsp12), non-structural protein 13 (Nsp13) helicase, and papain-like proteinase (PLpro) are molecules associated with SARS-CoV infection and propagation. SARS-CoV-2 can induce host cell death via five kinds of regulated cell death, i.e., apoptosis, necroptosis, pyroptosis, autophagy, and PANoptosis. The mechanisms of these cell deaths are well established and can be disrupted by synthetic small molecules or natural products. There are a variety of compounds proven to play roles in the cell death inhibition, such as pan-caspase inhibitor (z-VAD-fmk) for apoptosis, necrostatin-1 for necroptosis, MCC950, a potent and specific inhibitor of the NLRP3 inflammasome in pyroptosis, and chloroquine/hydroxychloroquine, which can mitigate the corresponding cell death pathways. However, NF-κB signaling is another critical anti-apoptotic or survival route mediated by SARS-CoV-2. Such signaling promotes viral survival, proliferation, and inflammation by inducing the expression of apoptosis inhibitors such as Bcl-2 and XIAP, as well as cytokines, e.g., TNF. As a result, tiny natural compounds functioning as proteasome inhibitors such as celastrol and curcumin can be used to modify NF-κB signaling, providing a responsible method for treating SARS-CoV-2-infected patients. The natural constituents that aid in inhibiting viral infection, progression, and amplification of coronaviruses are also emphasized, which are in the groups of alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones. Natural constituents derived from medicinal herbs have anti-inflammatory and antiviral properties, as well as inhibitory effects, on the viral life cycle, including viral entry, replication, assembly, and release of COVID-19 virions. The phytochemicals contain a high potential for COVID-19 treatment. As a result, SARS-CoV-2-infected cell death processes and signaling might be of high efficacy for therapeutic targeting effects and yielding encouraging outcomes.     

Dermal Tattoo Biosensors for Colorimetric Metabolite Detection

Tattooing is a ubiquitous body modification involving the injection of ink and/or dye pigments into the dermis. Biosensors in the form of tattoos can be used to monitor metabolites in interstitial fluid. Here, minimally invasive, injectable dermal biosensors were developed for measuring pH, glucose, and albumin concentrations. The dermal pH sensor was based on methyl red, bromothymol blue, and phenolphthalein, which responded to a pH range from 5.0 to 9.0. The dermal glucose sensor consisted of glucose oxidase, 3,3′,5,5′‐tetramethylbenzidine, and peroxidase that detected concentrations up to 50.0 mmol L^?1. The dermal albumin sensor consisted of 3′,3′′,5′,5′′‐tetrachlorophenol‐3,4,5,6‐tetrabromosulfophthalein to measure concentrations up to 5.0 g L^?1. The sensors were multiplexed in ex vivo skin tissue and quantitative readouts were obtained using a smartphone camera. These sensors can be used to manage of acid–base homeostasis, diabetes, and liver failure in point‐of‐care settings.     

Simultaneous Quantification of Heavy Metals Using a Solid State Potentiometric Sensor Array

A potentiometric sensor array of four nonspecific electrodes with solid‐state membranes is developed and tested for simultaneous analysis of copper(II), mercury(II), and silver(I) ions. The cross‐sensitivity responses of the sensors for these ions are evaluated. The array potentiometric signals are processed by partial least‐squares regression (PLS) and back propagation artificial neural networks (ANN) to determinate analyte concentrations. The ANN configuration is optimized and two different training algorithms of the ANN are also evaluated. Best results are obtained when the potentiometric sensors are activated and the data are processed using ANN and the gradient descent adaptive algorithm. The system is used to quantify these heavy metals in synthetic samples and in dental amalgams with successful results.