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The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand.
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Técnicas Biosensibles , ARN Polimerasas Dirigidas por ADN/ultraestructura , ADN/ultraestructura , Imagen Molecular/métodos , Nanotecnología/métodos , ARN/ultraestructura , Aptámeros de Nucleótidos/química , Emparejamiento Base , ADN/química , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Hibridación Fluorescente in Situ , Microscopía de Fuerza Atómica , Nanoestructuras/química , Nanotecnología/instrumentación , Conformación de Ácido Nucleico , ARN/química , Spinacia oleracea/químicaRESUMEN
Semipermeable membranes are a key feature of all living organisms. While specialized membrane transporters in cells can import otherwise impermeable nutrients, the earliest cells would have lacked a mechanism to import nutrients rapidly under nutrient-rich circumstances. Using both experiments and simulations, we find that a process akin to passive endocytosis can be recreated in model primitive cells. Molecules that are too impermeable to be absorbed can be taken up in a matter of seconds in an endocytic vesicle. The internalized cargo can then be slowly released over hours, into the main lumen or putative cytoplasm. This work demonstrates a way by which primitive life could have broken the symmetry of passive permeation prior to the evolution of protein transporters.
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Células Artificiales , Endocitosis , Vesículas TransportadorasRESUMEN
ConspectusDNA nanodevices are nanoscale assemblies, formed from a collection of synthetic DNA strands, that may perform artificial functions. The pioneering developments of a DNA cube by Nadrian Seeman in 1991 and a DNA nanomachine by Turberfield and Yurke in 2000 spawned an entire generation of DNA nanodevices ranging from minimalist to rococo architectures. Since our first demonstration in 2009 that a DNA nanodevice can function autonomously inside a living cell, it became clear that this molecular scaffold was well-placed to probe living systems. Its water solubility, biocompatibility, and engineerability to yield molecularly identical assemblies predisposed it to probe and program biology.Since DNA is a modular scaffold, one can integrate independent or interdependent functionalities onto a single assembly. Work from our group has established a new class of organelle-targeted, DNA-based fluorescent reporters. These reporters comprise three to four oligonucleotides that each display a specific motif or module with a specific function. Given the 1:1 stoichiometry of Watson-Crick-Franklin base pairing, all modules are present in a fixed ratio in every DNA nanodevice. These modules include an ion-sensitive dye or a detection module and a normalizing dye for ratiometry that along with detection module forms a "measuring module". The third module is an organelle-targeting module that engages a cognate protein so that the whole assembly is trafficked to the lumen of a target organelle. Together, these modules allow us to measure free ion concentrations with accuracies that were previously unattainable, in subcellular locations that were previously inaccessible, and at single organelle resolution. By revealing that organelles exist in different chemical states, DNA nanodevices are providing new insights into organelle biology. Further, the ability to deliver molecules with cell-type and organelle level precision in animal models is leading to biomedical applications.This Account outlines the development of DNA nanodevices as fluorescent reporters for chemically mapping or modulating organelle function in real time in living systems. We discuss the technical challenges of measuring ions within endomembrane organelles and show how the unique properties of DNA nanodevices enable organelle targeting and chemical mapping. Starting from the pioneering finding that an autonomous DNA nanodevice could map endolysosomal pH in cells, we chart the development of strategies to target organelles beyond the endolysosomal pathway and expanding chemical maps to include all the major ions in physiology, reactive species, enzyme activity, and voltage. We present a series of vignettes highlighting the new biology unlocked with each development, from the discovery of chemical heterogeneity in lysosomes to identifying the first protein importer of Ca2+ into lysosomes. Finally, we discuss the broader applicability of targeting DNA nanodevices organelle-specifically beyond just reporting ions, namely using DNA nanodevices to modulate organelle state, and thereby cell state, with potential therapeutic applications.
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ADN , Orgánulos , Orgánulos/química , Orgánulos/metabolismo , Humanos , ADN/química , Colorantes Fluorescentes/química , Animales , Nanoestructuras/químicaRESUMEN
Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.
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ADN/química , Lisosomas/metabolismo , Macrófagos/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Fagocitosis/fisiología , Animales , Aptámeros de Nucleótidos/farmacología , Autofagia/fisiología , Células COS , Calcio/metabolismo , Carbocianinas/farmacología , Línea Celular Tumoral , Chlorocebus aethiops , Células Hep G2 , Humanos , Lisosomas/efectos de los fármacos , Ratones , Nanocompuestos/química , Fagosomas/metabolismo , Células RAW 264.7RESUMEN
Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.
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Encéfalo/enzimología , ADN/química , Colorantes Fluorescentes/química , Óxido Nítrico Sintasa de Tipo II , Animales , Encéfalo/metabolismo , Química Encefálica , ADN/metabolismo , Colorantes Fluorescentes/metabolismo , Técnicas de Inactivación de Genes , Ratones , Microglía/química , Microglía/enzimología , Microglía/metabolismo , Microscopía Fluorescente , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Óxido Nítrico Sintasa de Tipo II/análisis , Óxido Nítrico Sintasa de Tipo II/química , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fagosomas/química , Fagosomas/metabolismo , Pez CebraRESUMEN
Impaired chloride transport affects diverse processes ranging from neuron excitability to water secretion, which underlie epilepsy and cystic fibrosis, respectively. The ability to image chloride fluxes with fluorescent probes has been essential for the investigation of the roles of chloride channels and transporters in health and disease. Therefore, developing effective fluorescent chloride reporters is critical to characterizing chloride transporters and discovering new ones. However, each chloride channel or transporter has a unique functional context that demands a suite of chloride probes with appropriate sensing characteristics. This Review seeks to juxtapose the biology of chloride transport with the chemistries underlying chloride sensors by exploring the various biological roles of chloride and highlighting the insights delivered by studies using chloride reporters. We then delineate the evolution of small-molecule sensors and genetically encoded chloride reporters. Finally, we analyze discussions with chloride biologists to identify the advantages and limitations of sensors in each biological context, as well as to recognize the key design challenges that must be overcome for developing the next generation of chloride sensors.
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Técnicas Biosensibles/métodos , Cloruros/metabolismo , HumanosRESUMEN
It is extremely challenging to quantitate lumenal Ca2+ in acidic Ca2+ stores of the cell because all Ca2+ indicators are pH sensitive, and Ca2+ transport is coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca2+ simultaneously, CalipHluor functions as a pH-correctable Ca2+ reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca2+ changes during endosomal maturation and found a surge in lumenal Ca2+ specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca2+ accumulation-an example of a lysosome-specific Ca2+ importer in animals. By enabling the facile quantification of compartmentalized Ca2+, CalipHluor can expand the understanding of subcellular Ca2+ importers.
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Calcio/metabolismo , ADN/química , Endosomas/metabolismo , Colorantes Fluorescentes/química , Concentración de Iones de Hidrógeno , Lisosomas/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Transporte Iónico , ATPasas de Translocación de Protón/metabolismo , Transducción de SeñalRESUMEN
The originally published paper has been updated to include the following new reference, added as ref. 18: Albrecht, T., Zhao, Y., Nguyen, T. H., Campbell, R. E. & Johnson, J. D. Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations. Cell Calcium 57, 263-274 (2015). Subsequent references have been renumbered in the reference list and throughout the text. Minor text changes were made in the sentence in which this new reference is first cited: "Previous attempts used endocytic tracers bearing either pH- or Ca2+-sensitive dyes to serially measure population-averaged pH and apparent Ca2+ in different batches of cells, thus scrambling information from individual endosomes13-17" in the original introduction was changed to "Previous attempts used endocytic tracers bearing either pH- or Ca2+-sensitive dyes13-17 or fluorescent-protein-based sensors18 to serially measure population-averaged pH and apparent Ca2+ in different batches of cells, thus scrambling information from individual endosomes." These changes have been made in the HTML and PDF versions of the article.
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Nitric oxide synthase 3 (NOS3) produces the gasotransmitter nitric oxide (NO), which drives critical cellular signaling pathways by S-nitrosylating target proteins. Endogenous NOS3 resides at two distinct subcellular locations: the plasma membrane and the trans-Golgi network (TGN). However, NO generation arising from the activities of both these pools of NOS3 and its relative contribution to physiology or disease is not yet resolvable. We describe a fluorescent DNA-based probe technology, NOckout, that can be targeted either to the plasma membrane or the TGN, where it can quantitatively map the activities of endogenous NOS3 at these locations in live cells. We found that, although NOS3 at the Golgi is tenfold less active than at the plasma membrane, its activity is essential for the structural integrity of the Golgi. The newfound ability to spatially map NOS3 activity provides a platform to discover selective regulators of the distinct pools of NOS3.
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ADN/química , Colorantes Fluorescentes/química , Óxido Nítrico Sintasa de Tipo III/metabolismo , Carbamatos/química , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Cinética , Óxido Nítrico/metabolismo , Imagen Óptica , Polietilenglicoles/química , Imagen Individual de Molécula , Red trans-Golgi/metabolismoRESUMEN
Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome-lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.
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ADN/química , Colorantes Fluorescentes/química , Ácido Hipocloroso/química , Fagosomas/química , Concentración de Iones de Hidrógeno , Oxidación-ReducciónRESUMEN
Membrane-initiated steroid signaling (MISS) is a recently discovered aspect of steroidal control over cell function that has proved highly challenging to study due to its rapidity and ultrasensitivity to the steroid trigger [Chow RWY, Handelsman DJ, Ng MKC (2010) Endocrinology 151:2411-2422]. Fundamental aspects underlying MISS, such as receptor binding, kinetics of ion-channel opening, and production of downstream effector molecules remain obscure because a pristine molecular technology that could trigger the release of signaling steroids was not available. We have recently described a prototype DNA nanocapsule which can be programmed to release small molecules upon photoirradiation [Veetil AT, et al. (2017) Nat Nanotechnol 12:1183-1189]. Here we show that this DNA-based molecular technology can now be programmed to chemically trigger MISS, significantly expanding its applicability to systems that are refractory to photoirradiation.
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Membrana Celular/metabolismo , ADN/química , Nanocápsulas/química , Transducción de Señal , Esteroides/metabolismo , Calcio/metabolismo , Células Cultivadas , Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Modelos Biológicos , Nanotecnología/métodos , Óxido Nítrico/metabolismo , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
As a ubiquitous second messenger, cyclic adenosine monophosphate (cAMP) mediates diverse biological processes such as cell growth, inflammation, and metabolism. The ability to probe these pathways would be significantly enhanced if we had a DNA-based sensor for cAMP. Herein, we describe a new, 31-base long single-stranded DNA aptamer for cAMP, denoted caDNApt-1, that was isolated by in vitro selection using systemic evolution of ligands after exponential enrichment (SELEX). caDNApt-1 has an approximately threefold higher affinity for cAMP than ATP, ADP, and AMP. Using non-denaturing gel electrophoresis and fluorescence spectroscopy, we characterized the structural changes caDNApt-1 undergoes upon binding to cAMP and revealed its potential as a cAMP sensor.
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Aptámeros de Nucleótidos/química , AMP Cíclico/análisis , Técnica SELEX de Producción de Aptámeros , Conformación de Ácido NucleicoRESUMEN
A family of asymmetric thiazolo[5,4-d]thiazole (TTz) fluorescent dye sensors has been developed, and their photophysical sensing properties are reported. The π-conjugated, TTz-bridged compounds are synthesized via a single-step, double condensation/oxidation of dithiooxamide and two different aromatic aldehydes: one with strong electron-donating characteristics and one with strong electron-accepting characteristics. The four reported dyes include electron-donating moieties (N,N-dibutylaniline and N,N-diphenylaniline) matched with three different electron-accepting moieties (pyridine, benzoic acid, and carboxaldehyde). The asymmetric TTz derivatives exhibit strong solvatofluorochromism with Stokes shifts between 0.269 and 0.750 eV (2270 and 6050 cm-1) and transition dipole moments (Δµ = 13-18 D) that are among the highest reported for push-pull dyes. Fluorescence quantum yields are as high as 0.93 in nonpolar solvents, and the fluorescence lifetimes (τF) vary from 1.50 to 3.01 ns depending on the solvent polarity. In addition, thermofluorochromic studies and spectrophotometric acid titrations were performed and indicate the possibility of using these dyes as temperature and/or acid sensors. In vitro cell studies indicate good cell membrane localization, negligible cytotoxicity, promising voltage sensitivities, and photostabilities that are 4 times higher than comparable dyes. Their ease of synthesis and purification, remarkable photophysical properties, and chemically sensitive TTz π-bridge make these asymmetric dye derivatives attractive for environmental and biological sensing or similar molecular optoelectronic applications.
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Bound calcium ions stabilize many nonenveloped virions. Loss of Ca2+ from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca2+-stabilized protein (VP7). Membrane-coupled conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double-layered inner capsid particle (DLP) into the cytosol. Removal of Ca2+in vitro strips away both outer layer proteins; we and others have postulated that the loss of Ca2+ triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca2+ sensor, the timing of Ca2+ loss from entering virions with respect to the dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer layer dissociation and DLP release. The Ca2+ sensor, placed on VP5*, monitors the Ca2+ concentration within the membrane-bound vesicle enclosing the entering particle. Slow (1-min duration) loss of Ca2+ precedes the onset of VP7 dissociation by about 2 min and DLP release by about 7 min. Coupled with our previous results showing that VP7 loss follows tight binding to the cell surface by about 5 min, these data indicate that Ca2+ loss begins as soon as the particle has become fully engulfed within the uptake vesicle. We discuss the implications of these findings for the molecular mechanism of membrane disruption during viral entry.IMPORTANCE Nonenveloped viruses penetrate into the cytosol of the cells that they infect by disrupting the membrane of an intracellular compartment. The molecular mechanisms of membrane disruption remain largely undefined. Functional reconstitution of infectious rotavirus particles (TLPs) from RNA-containing core particles (DLPs) and the outer layer proteins that deliver them into a cell makes these important pediatric pathogens particularly good models for studying nonenveloped virus entry. We report here how the use of a fluorescent Ca2+ sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca2+ loss from an entering particle and other molecular events in the entry pathway. Specific Ca2+ binding stabilizes many other viruses of eukaryotes, and Ca2+ loss appears to be a trigger for steps in penetration or uncoating. The experimental design that we describe may be useful for studying entry of other viral pathogens.
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Calcio/metabolismo , Proteínas de la Cápside/metabolismo , Colorantes Fluorescentes/química , Rotavirus/fisiología , Animales , Antígenos Virales/química , Antígenos Virales/metabolismo , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/química , Línea Celular , Citosol/virología , Microscopía Confocal , Conformación Proteica , Internalización del VirusRESUMEN
The miR-17-92a cluster, also known as 'oncomiR-1', is an RNA transcript that plays a pivotal regulatory role in cellular processes, including the cell cycle, proliferation and apoptosis. Its dysregulation underlies the development of several cancers. Oncomir-1 comprises six constituent miRNAs, each processed with different efficiencies as a function of both developmental time and tissue type. The structural mechanisms that regulate such differential processing are unknown, and this has impeded our understanding of the dysregulation of oncomiR-1 in pathophysiology. By probing the sensitivity of each nucleotide in oncomiR-1 to reactive small molecules, we present a secondary structural map of this RNA at single-nucleotide resolution. The secondary structure and solvent accessible regions of oncomiR-1 reveal that most of its primary microRNA domains are suboptimal substrates for Drosha-DGCR8, and therefore resistant to microprocessing. The structure indicates that the binding of trans-acting factors is required to remodel the tertiary organization and unmask cryptic primary microRNA domains to facilitate their processing into pre-microRNAs.
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MicroARNs/química , Humanos , Radical Hidroxilo/química , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Nucleótidos/química , Filogenia , Ribonucleasa III/metabolismo , Dispersión del Ángulo Pequeño , Ésteres del Ácido Sulfúrico/química , Termodinámica , Difracción de Rayos XRESUMEN
We describe a new method to measure viscosity within subcellular organelles of a living cell using nanorheology. We demonstrate proof of concept by measuring viscosity in lysosomes in multiple cell types and disease models. The lysosome is an organelle responsible for the breakdown of complex biomolecules. When different lysosomal proteins are defective, they are unable to break down specific biological substrates, which get stored within the lysosome, causing about 70 fatal diseases called lysosomal storage disorders (LSDs). Although the buildup of storage material is critical to the pathology of these diseases, methods to monitor cargo accumulation in the lysosome are lacking for most LSDs. Using passive particle tracking nanorheology and fluorescence recovery after photobleaching, we report that viscosity in the lysosome increases significantly during cargo accumulation in several LSD models. In a mammalian cell culture model of Niemann Pick C, lysosomal viscosity directly correlates with the levels of accumulated cholesterol. We also observed increased viscosity in diverse LSD models in Caenorhabditis elegans, revealing that lysosomal viscosity is a powerful reporter with which to monitor substrate accumulation in LSDs for new diagnostics or to assay therapeutic efficacy.
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Colorantes Fluorescentes/análisis , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/patología , Nanopartículas/análisis , Reología/métodos , Viscosidad , Animales , Caenorhabditis elegans , Línea Celular , Colesterol/análisis , Humanos , Lisosomas/química , Imagen Óptica/métodosRESUMEN
Extracellular DNA is engulfed by innate immune cells and digested by endosomal DNaseâ II to generate an immune response. Quantitative information on endosomal stage-specific cargo processing is a critical parameter to predict and model the innate immune response. Biochemical assays quantify endosomal processing but lack organelle-specific information, while fluorescence microscopy has provided the latter without the former. Herein, we report a single molecule counting method based on fluorescence imaging that quantitatively maps endosomal processing of cargo DNA in innate immune cells with organelle-specific resolution. Our studies reveal that endosomal DNA degradation occurs mainly in lysosomes and is negligible in late endosomes. This method can be used to study cargo processing in diverse endocytic pathways and measure stage-specific activity of processing factors in endosomes.