Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany.
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.
Methods Mol Biol. 2024;2822:87-100. doi: 10.1007/978-1-0716-3918-4_8.
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.
观察单个 RNA 分子可以深入了解它们的调控、与其他细胞成分的相互作用、组织和功能。尽管荧光点亮适体 (FLAPs) 最近在 RNA 成像方面显示出了前景，但由于其亮度和光稳定性差，其更广泛的应用受到了很大限制。我们最近开发了一种基于亲和力的 FLAP，称为 biRhoBAST，由于其出色的光稳定性和高亮度，它可以在活细胞或固定细胞中进行单分子 RNA 成像，并在活细胞中追踪单个 mRNA 分子。在这里，我们提供了从将 biRhoBAST 重复序列克隆到目标 RNA 序列开始的详细逐步协议，以对单个 mRNA 分子的动态成像。此外，我们还通过单分子荧光原位杂交 (smFISH) 和共定位研究来验证单分子成像实验。
