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A Live-Cell NanoBRET Assay to Monitor RNA-Protein Interactions and Their Inhibition by Small Molecules.
Jingsong Shan1,2, Amirhossein Taghavi2, Elizabeth A Caine3
1Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States.
ACS Central Science
|December 4, 2025
Summary
Researchers developed a live-cell assay to study RNA-protein interactions in myotonic dystrophy type 1 (DM1). This assay identified small molecules that disrupt toxic RNA-protein complexes and rescue DM1 cellular defects.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- RNA-protein interactions are vital for cellular functions but their disruption causes diseases like myotonic dystrophy type 1 (DM1).
- In DM1, expanded CUG repeats (r-(CUG)exp) sequester MBNL1, a splicing regulator, leading to aberrant pre-mRNA splicing and nuclear foci formation.
Purpose of the Study:
- To develop and validate a live-cell NanoBioluminescence Resonance Energy Transfer (NanoBRET) assay for studying RNA-protein interactions.
- To screen for small molecules that disrupt the interaction between r-(CUG)exp and MBNL1, a key event in DM1 pathogenesis.
- To assess the therapeutic potential of identified small molecules in rescuing DM1 cellular phenotypes.
Main Methods:
- A live-cell NanoBRET assay was established using MBNL1-NanoLuciferase and MBNL1-HaloTag constructs, with r-(CUG)exp serving as a scaffold.
- An RNA-focused small molecule library was screened using the NanoBRET assay to identify inhibitors of the r-(CUG)exp-MBNL1 complex.
- Nuclear magnetic resonance (NMR) was employed to determine the binding site of the identified inhibitors on r-(CUG)exp.
- Patient-derived myotubes were used to evaluate the efficacy of the small molecules in correcting DM1 cellular hallmarks.
Main Results:
- The NanoBRET assay successfully quantified RNA-protein interactions in live cells.
- Screening identified ten compounds with shared chemotypes that disrupt the r-(CUG)exp-MBNL1 complex.
- NMR studies revealed that these inhibitors bind to the 1x1 UU internal loops of folded r-(CUG)exp.
- Five of the identified molecules significantly rescued pre-mRNA splicing defects and reduced nuclear foci in DM1 patient-derived myotubes.
Conclusions:
- The live-cell NanoBRET assay is a powerful tool for investigating RNA-protein interactions and for drug discovery.
- Small molecules targeting the r-(CUG)exp-MBNL1 interaction can effectively ameliorate DM1 cellular pathology.
- This study provides a promising therapeutic strategy for DM1 by targeting specific RNA-protein interactions.
