Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose a concept of cell-permeable fluorogenic dimer of sulforhodamine B dyes (Gemini-561) and corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The unprecedented brightness and photostability together with high affinity of this complex allowed, for the first time, direct fluorescence imaging in live mammalian cells of RNA polymerase-III transcription products as well as messenger RNAs labelled with a single copy of the aptamer, i.e. without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
The inability to visualize single organic nanoparticles (NPs) in vivo is an unsolved issue in the field of nanomedicine. To enable high single particle fluorescence, we loaded polymer poly(methyl methacrylate)-sulfonate (PMMA-SO3H) NPs with octadecyl rhodamine B together with hydrophobic counter-ions as a fluorophore insulator to prevent aggregation-induced quenching. To create NPs with stealth properties we used the amphiphilic block copolymers pluronic F-127 and F-68. Fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) showed that pluronics remain at the NP surface after dialysis (density: one amphiphile per 5.5 nm 2) and protected NPs from degradation by serum proteins and surfactants. Single particle brightness was increased 150-fold as compared to commercially available preparations by increasing dye loading to 20 wt% and optimizing particle size. Added to primary cultured neurons, these NPs were stable and interacted with dendrites and axons. After intravenous injection in mice, NPs could be tracked in cerebral vessels for at least 1h by in vivo two-photon microscopy. Following brain injury or neuroinflammation, NPs extravasated from meningeal vessels, were taken up by meningeal macrophages, and entered the brain parenchyma. In summary, we developed biocompatible NPs suitable for drug delivery with in vitro and in vivo stealth properties and extremely bright fluorescence. Thus, individual NPs could be tracked in mouse brain and their dynamics visualized with subcellular resolution. Using superbright NPs may thus open new avenues for the investigation of NPs in living organisms.
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