Protein–DNA Chimeras for Nano Assembly

In synthetic biology, "understanding by building" requires exquisite control of the molecular constituents and their spatial organization. Site-specific coupling of DNA to proteins allows arrangement of different protein functionalities with emergent properties by self-assembly on origami-like DNA scaffolds or by direct assembly via Single-Molecule Cut & Paste (SMC&P). Here, we employed the ybbR-tag/Sfp system to covalently attach Coenzyme A-modified DNA to GFP and, as a proof of principle, arranged the chimera in different patterns by SMC&P. Fluorescence recordings of individual molecules proved that the proteins remained folded and fully functional throughout the assembly process. The high coupling efficiency and specificity as well as the negligible size (11 amino acids) of the ybbR-tag represent a mild, yet versatile, general and robust way of adding a freely programmable and highly selective attachment site to virtually any protein of interest.