Rational Design of a Multivalent RNA Combining Structural Motifs Tailored to Multiple Domains of Fused in Sarcoma for Potent Inhibition of Aggregation.

Fused in sarcoma (FUS) is an RNA-binding protein whose pathological aggregation, driven by aberrant phase separation, is implicated in amyotrophic lateral sclerosis (ALS). Although RNA molecules can modulate the FUS phase behavior, identifying highly effective sequences remains challenging because of FUS's multiple low-specificity RNA-binding domains. In this study, we rationally designed a 65-mer RNA, U1'+TERRA, by combining a stem-loop-GGU motif and a G-quadruplex (G4) structure, each known to interact with distinct FUS domains. U1'+TERRA exhibited strong binding affinity and effectively inhibited FUS aggregation in vitro. We introduced 2'-O-methyl modifications, generating (U1'+TERRA)-2'-OMe, which retained structural integrity and demonstrated resistance to nuclease degradation to enhance biological stability. Notably, (U1'+TERRA)-2'-OMe suppressed FUS aggregation even at a low concentration. These findings suggested that multivalent RNA constructs with rationally arranged motifs can serve as potent inhibitors of FUS aggregation. Our approach highlights the potential of structure-guided RNA engineering for the development of nucleic acid therapeutics targeting RNA-binding proteins involved in neurodegenerative diseases, such as ALS.