De novo design of Ras isoform selective binders

The proto-oncogene Ras which governs diverse intracellular pathways has four major isoforms (KRAS4A, KRAS4B, HRAS, and NRAS) with substantial sequence homology and similar in vitro biochemistry. There is considerable interest in investigating the roles of these independently as their association with different cancers vary, but there are few Ras isoform-specific binding reagents as the only significant sequence differences are in their disordered and highly charged C-termini which have been difficult to elicit antibodies against. To overcome this limitation, we use deep learning-based methods to de novo design Ras isoform-specific binders (RIBs) for KRAS4A, KRAS4B, and NRAS that specifically target the Ras C-terminus. The RIBs bind to their target Ras isoforms both in vitro and in cells with remarkable specificity, disrupted their membrane localization, and inhibited Ras activity. Therefore, these tools can contribute to dissecting the distinct roles of Ras isoforms in biology and disease.