Fluorogenic Interacting Protein Stabilization for Orthogonal RNA Imaging.

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Live RNA imaging is essential for understanding cellular functions.
• Existing genetically encodable RNA imaging systems face challenges in multiplexed and in vivo applications.

Purpose of the Study:

• To introduce a novel RNA imaging concept, Fluorogenic Interacting Protein Stabilization (FLIPS).
• To engineer RNA-binding proteins (RBPs) into orthogonal RNA-stabilized fluorogenic proteins for multiplexed RNA imaging.

Main Methods:

• Engineered fluorescence protein-fused RBPs (e.g., MCP, L7Ae, Cse3, LIN28A) using circular permutation.
• Incorporated a C-terminal poly(arginine)-appended degron into engineered RBPs.
• Leveraged RNA motif binding to stabilize engineered RBPs via proximity-mediated electrostatic interactions.

Main Results:

• Demonstrated orthogonal and multi-color fluorescence-activated RNA imaging.
• Successfully performed multicolor and orthogonal RNA imaging, single-molecule RNA imaging and tracking.
• Achieved simultaneous imaging of two RNAs in nuclear condensates and biplexed tracking of RNA translocation into cytosolic condensates.

Conclusions:

• The FLIPS system provides a versatile strategy for diverse RNA motifs and RBPs.
• This method enables advanced RNA imaging applications, including multiplexed and in vivo studies.
• Highlights potential for interrogating RNA biology and developing novel RNA-based imaging tools.