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


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.

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


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.

J Am Chem Soc ; 139(50): 18182-18185, 2017 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-29211468


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.

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