LAUSR

The hybrid binding domain (HBD) is a conserved fold present in ribonucleases H1 that selectively recognizes RNA–DNA hybrids, which are structures present in cellular R‐loops and participate in diverse biological processes. We engineered multivalent HBD proteins to create high‐affinity hybrid binders. Using EMSA‐ and SPR‐based analyses, we showed that the triple‐HBD protein exhibits a ~ 22 000‐fold increase in hybrid affinity (KD 370 pm) relative to the single HBD (KD 8.29 μm), with the length and sequence of the linkers enabling optimal function. These findings provide a framework for testing models that correlate multivalency and affinity to understand how multivalent proteins function and also can serve to guide applications that exploit multivalency as a strategy to enhance binding affinity.