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:
An RNA-based fluorogenic module consists of a light-up RNA aptamer able to specifically interact with a fluorogen to form a fluorescent complex. Over the past decade, significant efforts have been devoted to the development of such modules, which now cover the whole visible spectrum, as well as to their engineering to serve in a wide range of applications. In this review, we summarize the different strategies used to develop each partner (the fluorogen and the light-up RNA aptamer) prior to giving an overview of their applications that range from live-cell RNA imaging to the set-up of high-throughput drug screening pipelines. We then conclude with a critical discussion on the current limitations of these modules and how combining in vitro selection with screening approaches may help develop even better molecules.
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