RNA molecular recording with an engineered RNA deaminase

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• RNA molecular recorders are essential for studying RNA dynamics but are limited by RNA structure and sequence dependence.
• Existing tools like TRIBE, DART, and STAMP have inherent limitations affecting their efficiency and applicability.

Purpose of the Study:

• To develop an improved RNA molecular recording platform with enhanced activity, reduced bias, and lower background.
• To engineer a novel RNA adenosine base editor (rABE) for precise RNA base editing and molecular recording.
• To apply the developed platform for high-resolution mapping of RNA-protein interactions in single cells.

Main Methods:

• Directed evolution platform used to engineer a novel RNA adenosine base editor (rABE).
• Development of REMORA (RNA-encoded molecular recording in adenosines) utilizing the engineered rABE.
• Combination of rABE with APOBEC1 (C-to-U deaminase) and long-read RNA sequencing to measure RNA-binding protein interactions.

Main Results:

• Engineered rABE exhibits high activity, low bias, and low background, improving RNA molecular recording capabilities.
• REMORA successfully measured binding of two RNA-binding proteins (PUM1 and PUM2) on single messenger RNAs.
• Demonstrated competition between PUM1 and PUM2 for binding sites and identified transcript isoform-specific interactions.

Conclusions:

• The engineered rABE and REMORA platform overcome limitations of previous RNA molecular recorders.
• REMORA enables single-molecule, cell-type-specific identification of RNA-protein interactions.
• This technology provides novel insights into RNA regulation, including isoform-specific binding events.