RFOptimization: Guiding Design Optimization with All-Atom Structure Prediction

Abstract

Abstract Biomolecular interactions, including protein–protein interactions, protein–nucleic acid recognition, and protein–small molecule binding, underlie a wide range of biological processes and therapeutic mechanisms. Although recent de novo design methods can generate candidate binders for diverse molecular targets, practical design campaigns remain limited by low filter-passing rates and model-specific biases that arise when designs are optimized against a single predictor. Here, we present RFOptimization (RFO), a training-free framework for all-atom biomolecular binder optimization. RFO formulates binder improvement as a residue-wise mutational search problem, sampling candidate substitutions alternately using gradient-guided sequence optimization through all-atom structure prediction models and a cycling-based sequence redesign strategy that alternates structure generation with an orthogonal predictor and structure-conditioned inverse folding, thereby improving the in silico success rate of RFdiffusion-generated binders within minutes of computation. To reduce overfitting to any individual structure model, candidate mutations are further evaluated with orthogonal AlphaFold3 metrics as final filters. We demonstrate the generality of RFO across diverse design settings, including classical protein binder design, ligand-binding biosensor design, cyclic peptide design, and active site-aware enzyme design.