AI Article Synopsis
- Observing individual RNA molecules helps scientists understand how they work and interact within cells, but previous methods like fluorescent light-up aptamers (FLAPs) faced challenges with brightness and stability.
- The new biRhoBAST avidity-based FLAP improves this by enabling high-quality single-molecule RNA imaging in both live and fixed cells due to its superior brightness and photostability.
- This text outlines detailed protocols for using biRhoBAST to track mRNA dynamics and includes methods for validating these imaging experiments through techniques like smFISH and colocalization studies.
Article Abstract
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.
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| http://dx.doi.org/10.1007/978-1-0716-3918-4_8 | DOI Listing |
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