Jiang, B.; Li, X.; Guo, A.; Wei, M.; Wu, J.

The design of high-affinity protein binders is critical for biochemical detection, yet traditional methods remain labor-intensive. AI-driven tools like RFdiffusion, a RoseTTAFold-based diffusion model, offer promising alternatives for generating protein structures with tailored binding interfaces. This study evaluates RFdiffusion\'s efficacy in desinging de novo binders for six targets: Strep-TagII (a peptide tag) and five eukaryotic proteins (STAT3,FGF4,EGF,PDGF-BB and CD4). Five binders were designed for each target and experimentally validated. While two Strep-TagII binders outperformed streptavidin in Western blot assays, none matched the sensitivity of anti-Strep-TagII antibodies. Binders for the other targets failed due to low expression, nonspecific binding, or undetectable affinity. Despite generating structurally diverse candidates, RFdiffusion\'s success rate was limited by low-affinity designs and inconsistent recombinant expression. These results underscore the need for further optimization of AI-driven protein design tools for practical biochemical applications.