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Evolution Driven Microscale Combinatorial Chemistry in Intracellular Mimicking Droplets to Engineer Thermostable RNA
Andrew Brian Kinghorn1, Wei Guo2,3, Lin Wang1,3
1School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
Small (Weinheim an Der Bergstrasse, Germany)
|January 27, 2025
Summary
Researchers developed a novel biomimetic system using heated water droplets to evolve RNA aptamers with enhanced fluorescence and stability. The engineered eBroccoli aptamer improves cellular RNA tracking and visualization of stress granules.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Fluorescent aptamers are crucial for tracking RNA in cells but face limitations in stability and fluorescence.
- Current in vitro evolution methods fail to replicate complex intracellular environments, hindering in vivo applications.
- RNA folding is influenced by cellular microenvironments, a factor not well-simulated in traditional selection techniques.
Purpose of the Study:
- To create a biomimetic system that accurately simulates intracellular conditions for RNA evolution.
- To engineer novel RNA aptamers with improved fluorescence intensity and thermal stability for in vivo applications.
- To develop a platform for enhanced biomolecule engineering by mimicking cellular complexity.
Main Methods:
- Utilized microscale heated water droplets to mimic intracellular conditions and RNA folding landscapes.
- Integrated this system with microfluidic droplet sorting for efficient RNA aptamer evolution.
- Applied the platform to evolve a new RNA aptamer, named eBroccoli, under biomimetic conditions.
Main Results:
- Engineered the eBroccoli RNA aptamer with significantly enhanced fluorescence intensity (3.9-fold in mammalian cells) and thermal stability.
- Demonstrated in vitro and in vivo functionality in both bacterial and mammalian cells, with thermal stability up to 45°C in bacteria.
- Successfully visualized nanoscale stress granule formation in mammalian cells during heat shock using eBroccoli.
Conclusions:
- The biomimetic droplet system effectively replicates intracellular complexity, enabling the evolution of superior RNA aptamers.
- eBroccoli represents a significant advancement in fluorescent RNA tracking tools, offering improved performance in diverse cellular contexts.
- The concept of 'biomimetic equivalence' provides a powerful strategy for engineering biomolecules tailored to specific cellular environments.
