Live‐Cell Imaging of Endogenous mRNAs with a Small Molecule

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non‐engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene‐specific RNA aptamer, combined with a cell‐permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live‐cell imaging of the endogenous mRNA of β‐actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin‐2, cortactin, and cytoplasmic FMR1‐interacting protein 2. The RNA‐imaging technology and its further optimization might permit live‐cell imaging of any RNA molecules.     

Modifying the 5′‐Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells

The 5′‐cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5′‐cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7‐modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N²‐modified 5′‐caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live‐cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox.     

Tracking the Dynamic Folding and Unfolding of RNA G‐Quadruplexes in Live Cells

Because of the absence of methods for tracking RNA G‐quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G‐quadruplexes is so far limited. Herein, we report a new red‐emitting fluorescent probe, QUMA‐1 , for the selective, continuous, and real‐time visualization of RNA G‐quadruplexes in live cells. The applications of QUMA‐1 in several previously intractable applications, including live‐cell imaging of the dynamic folding, unfolding, and movement of RNA G‐quadruplexes and the visualization of the unwinding of RNA G‐quadruplexes by RNA helicase have been demonstrated. Notably, our real‐time results revealed the complexity of the dynamics of RNA G‐quadruplexes in live cells. We anticipate that the further application of QUMA‐1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G‐quadruplexes will uncover more information about the biological roles of RNA G‐quadruplexes.     

Inhibition of translation by small RNA-stabilized mRNA structures in human cells

RNA-mediated gene regulation and expression are critically dependent on both nucleic acid architecture and recognition. We present a novel mechanism for the regulation of gene expression through direct RNA-RNA interactions between small RNA and mRNA in human cells. Using mRNA reporters containing G-rich sequences in the 5'-untranslated region (5'-UTR), in the coding region, or both, we showed that G-rich small RNAs bind to the reporter mRNAs and form an intermolecular RNA G-quadruplex that can inhibit gene translation in living cells. Using a combination of circular dichroism (CD) and RNase footprinting in vitro, we found that the intermolecular G-quadruplexes show a parallel G-quadruplex structure. We next investigated whether the intermolecular G-quadruplex is present in living cells. Employing the fluorophore-labeled probes, we found that two G-rich RNA molecules form an intermolecular G-quadruplex structure in living cells. These results extend the concept of small RNA-mediated expression and suggest an important role for such RNA structures in the inhibition of mRNA translation.     

Development of a Highly Selective and Sensitive Fluorescent Probe for Imaging RNA Dynamics in Live Cells

RNA imaging is of great importance for understanding its complex spatiotemporal dynamics and cellular functions. Considerable effort has been devoted to the development of small-molecule fluorescent probes for RNA imaging. However, most of the reported studies have mainly focused on improving the photostability, permeability, long emission wavelength, and compatibility with live-cell imaging of RNA probes. Less attention has been paid to the selectivity and detection limit of this class of probes. Highly selective and sensitive RNA probes are still rarely available. In this study, a new set of styryl probes were designed and synthesized, with the aim of upgrading the detection limit and maintaining the selectivity of a lead probe QUID−1 for RNA. Among these newly synthesized compounds, QUID−2 was the most promising candidate. The limit of detection (LOD) value of QUID−2 for the RNA was up to 1.8 ng/mL in solution. This property was significantly improved in comparison with that of QUID−1. Further spectroscopy and cell imaging studies demonstrated the advantages of QUID−2 over a commercially available RNA staining probe, SYTO RNASelect, for highly selective and sensitive RNA imaging. In addition, QUID−2 exhibited excellent photostability and low cytotoxicity. Using QUID−2, the global dynamics of RNA were revealed in live cells. More importantly, QUID−2 was found to be potentially applicable for detecting RNA granules in live cells. Collectively, our work provides an ideal probe for RNA imaging. We anticipate that this powerful tool may create new opportunities to investigate the underlying roles of RNA and RNA granules in live cells.     

单病毒示踪

病毒是对人类健康威胁最大的病原之一,由其引发的病毒性疾病对人民健康、国家安全和社会经济构成重大威胁.病毒感染机制研究对病毒性疾病的防控及治疗具有重大意义.病毒侵染宿主细胞的动态过程涉及病毒组分与多种细胞组分或细胞器间复杂的相互作用,但是传统手段无法对该动态过程进行实时跟踪研究.单病毒示踪技术作为一种可以实时原位示踪单颗...     

High-contrast and real-time visualization of membrane proteins in live cells with malachite green-based fluorogenic probes

Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green(MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or noncatalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670...     

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.     

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.     

Functional Nanoparticles with a Reducible Tetrasulfide Motif to Upregulate mRNA Translation and Enhance Transfection in Hard-to-Transfect Cells

Effective messenger RNA (mRNA) transfection in hard-to-transfect cells delivered by vectors is a long-standing challenge. Now it is hypothesized that the high intracellular glutathione level is associated with suppressed mRNA translation. This theory leads to a new design principle of next-generation mRNA vectors: nanoparticles with glutathione depletion chemistry upregulate mRNA translation and enhance transfection, which is beneficial for mRNA delivery in hard-to-transfect cells in vitro and in vivo.     

Functional Nanoparticles with a Reducible Tetrasulfide Motif to Upregulate mRNA Translation and Enhance Transfection in Hard‐to‐Transfect Cells

Effective messenger RNA (mRNA) transfection in hard‐to‐transfect cells delivered by vectors is a long‐standing challenge. Now it is hypothesized that the high intracellular glutathione level is associated with suppressed mRNA translation. This theory leads to a new design principle of next‐generation mRNA vectors: nanoparticles with glutathione depletion chemistry upregulate mRNA translation and enhance transfection, which is beneficial for mRNA delivery in hard‐to‐transfect cells in vitro and in vivo.     

In Cellulo Generation of Fluorescent Probes for Live-Cell Imaging of Cylooxygenase-2

Live-cell imaging with fluorescent probes is an essential tool in chemical biology to visualize the dynamics of biological processes in real-time. Intracellular disease biomarker imaging remains a formidable challenge due to the intrinsic limitations of conventional fluorescent probes and the complex nature of cells. This work reports the in cellulo assembly of a fluorescent probe to image cyclooxygenase-2 (COX-2). We developed celecoxib-azide derivative 14 , possessing favorable biophysical properties and excellent COX-2 selectivity profile. In cellulo strain-promoted fluorogenic click chemistry of COX-2-engaged compound 14 with non/weakly-fluorescent compounds 11 and 17 formed fluorescent probes 15 and 18 for the detection of COX-2 in living cells. Competitive binding studies, biophysical, and comprehensive computational analyses were used to describe protein-ligand interactions. The reported new chemical toolbox enables precise visualization and tracking of COX-2 in live cells with superior sensitivity in the visible range.