Single-Molecule RNA Imaging in Live Cells with an Avidity-Based Fluorescent Light-Up Aptamer biRhoBAST.
| Autor: | Bühler B; Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany., Sunbul M; Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany. msunbul@uni-heidelberg.de. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2822, pp. 87-100. |
| DOI: | 10.1007/978-1-0716-3918-4_8 |
| Abstrakt: | Observing individual RNA molecules provides valuable insights into their regulation, interactions with other cellular components, organization, and functions. Although fluorescent light-up aptamers (FLAPs) have recently shown promise for RNA imaging, their wider applications have been mostly hindered by poor brightness and photostability. We recently developed an avidity-based FLAP known as biRhoBAST that allows for single-molecule RNA imaging in live or fixed cells and tracking individual mRNA molecules in living cells due to its excellent photostability and high brightness. Here, we present step-by-step detailed protocols starting from cloning biRhoBAST repeats into the target RNA sequence, to imaging dynamics of single mRNA molecules. Additionally, we address the validation of single-molecule imaging experiments through single-molecule fluorescence in situ hybridization (smFISH) and colocalization studies. (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Databáze: | MEDLINE |
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