Rapid Selection of RNA Aptamers that Activate Fluorescence of Small Molecules
RNA aptamers can serve as valuable tools for studying and manipulating live cells. Fluorescent aptamers are the ones that bind to and turn on fluorescence of small-molecule dyes (fluorogens). Similarly to fluorescent proteins, fluorescent RNA aptamers can be used to image spatial and temporal RNA dynamics in live cells. Additionally, these aptamers can serve as a basis for engineering genetically encoded fluorescent biosensors. This chapter presents a protocol for rapid and efficient screening of RNA aptamer libraries to isolate fluorescent aptamers. The protocol describes how to design, clone, and express RNA aptamer library in bacterial cells and how to screen the bacteria to find aptamers with the desired fluorescent properties.
Key wordsRNA Aptamer Fluorescence High-throughput screening Directed evolution FACS
The author thanks Prof. Samie R. Jaffrey for his generous support and scientific guidance. Flow cytometry experiments were performed with the help of J. McCormick and S.Z. Merlin (Department of Pathology and Laboratory Medicine cell sorter core). The author is also grateful to the members of the Jaffrey lab for their feedback as they used this protocol. This work was supported by NIH grants to Prof. Samie R. Jaffrey (R01 NS064516 and R01 EB010249).
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