Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Directly imaging biologically important RNAs in cells is challenging due to a lack of suitable fluorescent tools.
• Existing protein imaging tools, like fluorescent proteins, are not directly applicable to RNA studies.
• Ideal RNA labeling systems require high affinity, preservation of biological function, and favorable photophysical properties.

Purpose of the Study:

• To develop novel fluorescent aptamers for high-resolution RNA imaging in cellular environments.
• To create RNA labeling tools with photophysical properties comparable to existing fluorescent proteins.
• To enable the study of RNA localization and dynamics in live and fixed mammalian cells.

Main Methods:

• Microfluidics-based selection was employed to identify high-affinity RNA Mango fluorogenic aptamers.
• The photophysical properties of the new aptamers were characterized, including brightness compared to enhanced GFP.
• The aptamers were used to visualize the subcellular localization of specific small non-coding RNAs in mammalian cells.

Main Results:

• Three new high-affinity RNA Mango fluorogenic aptamers were successfully selected.
• Two of the developed aptamers demonstrated brightness equal to or exceeding enhanced GFP when bound to TO1-Biotin.
• Accurate imaging of subcellular localization for 5S, U6, and box C/D scaRNA was achieved in both live and fixed mammalian cells.

Conclusions:

• The newly developed RNA Mango aptamers offer a significant advancement for RNA visualization in biological systems.
• These aptamers provide a powerful new tool for studying RNA function, dynamics, and localization in vitro and in mammalian cells.
• The developed tools are compatible with established live and fixed cell protein labeling strategies, facilitating multi-modal imaging.