2Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain

Emerging DNA-based nanotechnologies would benefit from the ability to modulate the properties (e.g., solubility, melting temperature, chemical stability) of diverse DNA templates (single molecules or origami nanostructures) through controlled, self-assembling coatings. We here introduce a DNA coating agent, called C-8-B-Ss07d, which binds to and coats with high specificity and affinity, individual DNA molecules as well as folded origami nanostructures. C-8-B-Ss07d coats and protects without condensing, collapsing or destroying the spatial structure of the underlying DNA template. C-8-B-Ss07d combines the specific nonelectrostatic DNA binding affinity of an archeal-derived DNA binding domain (Sso7d, 7 kDa) with a long hydrophilic random coil polypeptide (C-8, 73 kDa), which provides colloidal stability (solubility) through formation of polymer brushes around the DNA templates. C-8-B-Ss07d is produced recombinantly in yeast and has a precise (but engineerable) amino acid sequence of precise length. Using electrophoresis, AFM, and fluorescence microscopy we demonstrate protein coat formation with stiffening of one-dimensional templates (linear dsDNA, supercoiled dsDNA and circular ssDNA), as well as coat formation without any structural distortion or disruption of two-dimensional DNA origami template. Combining the programmability of DNA with the nonperturbing precise coating capability of the engineered protein C8-B-Ss07d holds promise for future applications such as the creation of DNA-protein hybrid networks, or the efficient transfection of individual DNA nanostructures into cells.