A protein adaptor to locate a functional protein dimer on molecular switchboard

The addressable DNA nanostructures offer ideal platforms to construct organized assemblies of multiple protein molecules. Sequence-specific DNA binding proteins that target defined sites on DNA nanostructures would act as orthogonal adaptors to carry individual protein molecules to the programmed addresses. We have recently developed a protein-based adaptor by utilizing the sequence-specific DNA binding zinc finger protein to locate a monomeric protein of interest at specific positions on DNA origami, which serves as a molecular switchboard. We herein report a new adaptor to locate a protein dimer on the DNA origami scaffold based on a homodimeric basic-leucine zipper protein GCN4. Specific binding of GCN4 to programmed addresses on DNA origami and orthogonal targeting by GCN4- and zinc finger protein-based adaptors to the respective addresses on DNA origami were confirmed by gel electrophoretic and AFM analyses. Furthermore, a GCN4-fused homodimeric enzyme showed even higher activity than the wild type enzyme, and exhibited avid reactivity when assembled at the specific site of DNA origami. Thus, GCN4 serves as an ideal adaptor to locate homodimeric proteins in the functional form on DNA origami.