Covalent and Oriented Immobilization of Antibodies through Systematic Modification of Photoactivatable RNA Hybrid Aptamers

We report a covalent and well-oriented strategy of immobilization of antibodies using photoactivatable Fc-binding RNA aptamers (FcBAs). We prepared several types of FcBAs that were further modified with photoaffinity probes (i.e., benzophenone or diazirine), and evaluated the binding capabilities and the photo-crosslinking efficiency of them via pull-down assays and fluorescence analyses, respectively. Initial moderate photo-crosslinking efficiency (K-d similar to 110 nM) was substantially improved by multivalent association of FcBAs and structural modification of FcBAs. For a conceptual proof, covalent crosslinking of human IgG on FcBA-tethered solid chips was fabricated and investigated by scanometry, which eventually proved real applicability of the present scheme toward immunoassays.

Automated summary

We reported a strategy of immobilizing antibodies using photoactivatable Fc-binding RNA aptamers (FcBAs) in a covalent and well-oriented manner. We prepared modified FcBAs with photoaffinity probes and evaluated their binding capabilities and photo-crosslinking efficiency. The initial efficiency was improved by multivalent association and structural modification of FcBAs. We demonstrated the applicability of this strategy in immunoassays through the fabrication and investigation of covalent crosslinking on solid chips.