1. Evolution Driven Microscale Combinatorial Chemistry in Intracellular Mimicking Droplets to Engineer Thermostable RNA for Cellular Imaging.
- Author
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Kinghorn AB, Guo W, Wang L, Tang MYH, Wang F, Shiu SC, Lau KK, Jinata C, Poonam AD, Shum HC, and Tanner JA
- Subjects
- Combinatorial Chemistry Techniques methods, Aptamers, Nucleotide chemistry, Humans, Temperature, Intracellular Space metabolism, RNA chemistry
- Abstract
Fluorescent light-up aptamer/fluorogen pairs are powerful tools for tracking RNA in the cell, however limitations in thermostability and fluorescence intensity exist. Current in vitro selection techniques struggle to mimic complex intracellular environments, limiting in vivo biomolecule functionality. Taking inspiration from microenvironment-dependent RNA folding observed in cells and organelle-mimicking droplets, an efficient system is created that uses microscale heated water droplets to simulate intracellular conditions, effectively replicating the intracellular RNA folding landscape. This system is integrated with microfluidic droplet sorting to evolve RNA aptamers. Through this approach, an RNA aptamer is engineered with improved fluorescence activity by exploring the chemical fitness landscape under biomimetic conditions. The enhanced RNA aptamer named eBroccoli has increased fluorescence intensity and thermal stability, both in vitro and in vivo in bacterial and mammalian cells. In mammalian cell culture conditions, a fluorescence improvement of 3.9-times is observed and biological thermal stability up to 45 °C is observed in bacterial systems. eBroccoli enable real-time visualization of nanoscale stress granule formation in mammalian cells during heat shock at 42 °C. By introducing the concept of "biomimetic equivalence" based on RNA folding, the platform offers a simple yet effective strategy to mimic intracellular complexity in evolution-based engineering., (© 2025 The Author(s). Small published by Wiley‐VCH GmbH.)
- Published
- 2025
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