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.
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