• A novel ssDNA chimera approach enables characterization of RBP-RBP interactions under RNase treatment, distinguishing direct from RNA-mediated associations.
• The method successfully rescues RBP associations in the presence of RNase, demonstrating the active role of specific RNA sequences in mediating protein interactions.
• This technique provides a tool to study the molecular mechanisms underlying RBP aggregation and sequestration, relevant to neurodegenerative disease pathologies.
• The ssDNA chimeras are resistant to nuclease degradation, offering a stable alternative to RNA for probing RBP interactions in vitro.