Herein, we report the synthesis and isolation of a shape-persistent Janus protein nanoparticle derived from biomolecular machine chaperonin GroEL (AGroELB) and its application to DNA-mediated ternary supramolecular copolymerization. For synthesizing AGroELB with two different DNA strands A and B at its opposite apical domains, we utilized the unique biological property of GroEL, i.e., "Mg2+/ATP-mediated ring exchange" between AGroELA and BGroELB with their hollow cylindrical double-decker architectures. This exchange event was reported more than 24 years ago but has never been utilized for molecular engineering of GroEL. We leveraged DNA nanotechnology to purely isolate Janus AGroELB and succeeded in its precision ternary supramolecular copolymerization with two DNA comonomers A** and B* that are partially complementary to A and B in AGroELB, respectively, and programmed to self-dimerize on the other side. Transmission electron microscopy allowed us to confirm the formation of an expected dual-periodic copolymer sequence -(B*/BGroELA/A**/A**/AGroELB/B*)- in the form of a laterally connected lamellar assembly, rather than a single-chain copolymer.
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