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1.
J Am Chem Soc ; 142(6): 2968-2974, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31968164

RESUMO

In situ amplification methods, such as hybridization chain reaction, are valuable tools for mapping the spatial distribution and subcellular location of target analytes. However, the live-cell applications of these methods are still limited due to challenges in the probe delivery, degradation, and cytotoxicity. Herein, we report a novel genetically encoded in situ amplification method to noninvasively image the subcellular location of RNA targets in living cells. In our system, a fluorogenic RNA reporter, Broccoli, was split into two nonfluorescent fragments and conjugated to the end of two RNA hairpin strands. The binding of one target RNA can then trigger a cascaded hybridization between these hairpin pairs and thus activate multiple Broccoli fluorescence signals. We have shown that such an in situ amplified strategy can be used for the sensitive detection and location imaging of various RNA targets in living bacterial and mammalian cells. This new design principle provides an effective and versatile platform for tracking various intracellular analytes.

2.
Angew Chem Int Ed Engl ; 58(50): 18271-18275, 2019 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-31591798

RESUMO

Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology. Fluorogenic RNA-based sensors have emerged to detect various targets in living cells. However, it is still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using a pair of orthogonal fluorogenic RNA aptamers, DNB and Broccoli, we engineered a modular sensor system to apply the DNB-to-Broccoli fluorescence ratio to quantify the cell-to-cell variations of target concentrations. These ratiometric sensors can be broadly applied for live-cell imaging and quantification of metabolites, signaling molecules, and other synthetic compounds.

3.
Cell Chem Biol ; 26(4): 471-481.e3, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30773480

RESUMO

Genetically encoded biosensors are useful tools for detecting the presence and levels of diverse biomolecules in living cells. However, low-abundance targets are difficult to detect because they are often unable to bind and activate enough biosensors to detect using standard microscopic imaging approaches. Here we describe a type of RNA-based biosensor, an RNA integrator, which enables detection of low-abundance targets in vitro and in living cells. The RNA integrator is an RNA sequence comprising a ribozyme and an unfolded form of the fluorogenic aptamer Broccoli. Upon binding its target, the ribozyme undergoes cleavage and releases Broccoli, which subsequently folds and becomes fluorescent. Importantly, each target molecule can bind and induce cleavage of multiple copies of the integrator sensor, resulting in an amplified signal. We show that this approach can be generalized to numerous different ribozyme types for the detection of various small molecules.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , RNA Catalítico/química , Sequência de Bases , Escherichia coli/citologia , Imagem Óptica/métodos , Dobramento de RNA
4.
Methods ; 161: 24-34, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30660865

RESUMO

A fluorogenic aptamer can specifically interact with a fluorophore to activate its fluorescence. These nucleic acid-based fluorogenic modules have been dramatically developed over the past decade, and have been used as versatile reporters in the sensor development and for intracellular imaging. In this review, we summarize the design principles, applications, and challenges of the first-generation fluorogenic RNA-based sensors. Moreover, we discuss some strategies to develop next-generation biosensors with improved sensitivity, selectivity, quantification property, and eukaryotic robustness. Using genetically encoded catalytic hairpin assembly strategy as an example, we further introduce a standard protocol to design, characterize, and apply these fluorogenic RNA-based sensors for in vitro detection and cellular imaging of target biomolecules. By incorporating natural RNA machineries, nucleic acid nanotechnology, and systematic evolution approaches, next-generation fluorogenic RNA-based devices can be potentially engineered to be widely applied in cell biology and biomedicine.

5.
Chem Commun (Camb) ; 55(5): 707-710, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30566125

RESUMO

Silver has been widely used for disinfection. The cellular accumulation of silver ions (Ag+) is critical in these antibacterial effects. The direct cellular measurement of Ag+ is useful for the study of disinfection mechanisms. Herein, we reported a novel genetically encoded RNA-based sensor to image Ag+ in live bacterial cells. The sensor is designed by introducing a cytosine-Ag+-cytosine metallo base pair into a fluorogenic RNA aptamer, Broccoli. The binding of Ag+ induces the folding of Broccoli and activates a fluorescence signal. This sensor can be genetically encoded to measure the cellular flux and antibacterial effect of Ag+.


Assuntos
Antibacterianos/análise , Cátions Monovalentes/análise , Prata/análise , Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases/efeitos dos fármacos , Cátions Monovalentes/farmacologia , Nucleotídeos de Citosina/genética , Liberação Controlada de Fármacos , Escherichia coli/efeitos dos fármacos , Fluorescência , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Microscopia Confocal , Microscopia de Fluorescência , Conformação de Ácido Nucleico/efeitos dos fármacos , Prata/química , Prata/farmacologia
6.
J Am Chem Soc ; 140(28): 8739-8745, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-29944357

RESUMO

DNA and RNA nanotechnology has been used for the development of dynamic molecular devices. In particular, programmable enzyme-free nucleic acid circuits, such as catalytic hairpin assembly, have been demonstrated as useful tools for bioanalysis and to scale up system complexity to an extent beyond current cellular genetic circuits. However, the intracellular functions of most synthetic nucleic acid circuits have been hindered by challenges in the biological delivery and degradation. On the other hand, genetically encoded and transcribed RNA circuits emerge as alternative powerful tools for long-term embedded cellular analysis and regulation. Herein, we reported a genetically encoded RNA-based catalytic hairpin assembly circuit for sensitive RNA imaging inside living cells. The split version of Broccoli, a fluorogenic RNA aptamer, was used as the reporter. One target RNA can catalytically trigger the fluorescence from tens-to-hundreds of Broccoli. As a result, target RNAs can be sensitively detected. We have further engineered our circuit to allow easy programming to image various target RNA sequences. This design principle opens the arena for developing a large variety of genetically encoded RNA circuits for cellular applications.


Assuntos
Aptâmeros de Nucleotídeos/química , Escherichia coli/citologia , Corantes Fluorescentes/química , Imagem Óptica/métodos , RNA/análise , Aptâmeros de Nucleotídeos/genética , Escherichia coli/química , Escherichia coli/genética , Fluorescência , Conformação de Ácido Nucleico , RNA/genética , Espectrometria de Fluorescência/métodos
7.
J Am Chem Soc ; 139(50): 18182-18185, 2017 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-29211468

RESUMO

Mechanical forces play critical roles in collective cell behaviors such as cell migration, proliferation, and differentiation. Extensive efforts have been made to measure forces between cells and extracellular matrices. However, force studies at cell-cell junctions remain a challenge. Herein, we reported a novel strategy to construct membrane DNA tension probes to visualize tensile forces at cell junctions. These lipid-modified probes can self-assemble onto cell membranes with high efficiency and stability. Upon experiencing tensile forces generated by neighboring cells, unfolding of the probes leads to a large increase in the fluorescence intensity. Compatible with readily accessible fluorescence microscopes, these easy-to-use membrane DNA tension probes can be broadly used to measure intercellular tensile forces.


Assuntos
Sondas de DNA/química , DNA/química , Sondas Moleculares/química , Células 3T3 , Animais , Fenômenos Mecânicos , Camundongos , Modelos Biológicos
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