A multivalent RNA affinity tag enables selective purification of tagged RNAs and bound proteins.

Efficient purification of specific RNAs from lysates remains a major challenge. Existing methods, such as MS2 tagging or oligonucleotide hybridization, require protein immobilization or sequence-specific hybridization, limiting scalability and compatibility with diverse RNAs. Current approaches often have low recovery, exhibit slow kinetics, and generate background contamination, limiting their use in RNA-protein interaction studies. To overcome these limitations, we developed FS2, an RNA sequence that binds Sephadex beads with high affinity, thus acting as an affinity tag for protein-free, rapid, and simple RNA purification. FS2 consists of two copies of the dextran-binding D8 aptamer embedded within the highly stable F30 three-way junction RNA scaffold, which promotes aptamer folding and enhances avidity. Systematic optimization revealed that FS2 exhibits rapid binding kinetics, efficient purification, and efficient elution under mild conditions (50°C, 10 mM EDTA), representing substantial improvements over existing RNA purification systems. We validated the utility of FS2 by purifying an FS2-tagged MYC mRNA fragment from HEK293T cells and demonstrating that N6-methyladenosine (m6A)-containing mRNAs can be pulled down along with the m6A-binding protein YTHDF2. Overall, FS2 provides a highly simple and efficient RNA-based affinity tag for recovering RNA from complex mixtures and lysates for diverse applications.