Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, Strasbourg, France.
Université de Strasbourg, Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Illkirch, France.
Nat Chem Biol. 2020 Jan;16(1):69-76. doi: 10.1038/s41589-019-0381-8. Epub 2019 Oct 21.
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 the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, 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.
活细胞内 RNA 的成像一直是个挑战,因为缺乏天然荧光 RNA。最近开发的 RNA 适体,可以点亮小分子荧光染料,克服了这一限制,但它们仍然存在亮度和光稳定性差的问题。在这里,我们提出了一种细胞通透性的磺酰罗丹明 B 染料的荧光双联体(Gemini-561)的概念,以及相应的二聚体适体(o-Coral),它可以显著提高当前 RNA 成像方法的性能。该复合物具有较高的亲和力和改进的亮度和光稳定性,使得 RNA 聚合酶 III 转录产物以及带有单个适体拷贝的信使 RNA 可以直接在活哺乳动物细胞中进行荧光成像,而无需标签的多聚化。所开发的荧光模块可以实现活细胞内 RNA 的快速灵敏检测,而提出的设计概念为新一代超亮 RNA 探针开辟了道路。