Single molecule DNA origami nanoarrays with controlled protein orientation

The nanoscale organization of functional (bio)molecules on solid substrates with nanoscale spatial resolution and single-molecule control-in both position and orientation-is of great interest for the development of next-generation (bio)molecular devices and assays. Herein, we report the fabrication of nanoarrays of individual proteins (and dyes) via the selective organization of DNA origami on nanopatterned surfaces and with controlled protein orientation. Nanoapertures in metal-coated glass substrates were patterned using focused ion beam lithography; 88% of the nanoapertures allowed immobilization of functionalized DNA origami structures. Photobleaching experiments of dye-functionalized DNA nanostructures indicated that 85% of the nanoapertures contain a single origami unit, with only 3% exhibiting double occupancy. Using a reprogrammed genetic code to engineer into a protein new chemistry to allow residue-specific linkage to an addressable ssDNA unit, we assembled orientation-controlled proteins functionalized to DNA origami structures; these were then organized in the arrays and exhibited single molecule traces. This strategy is of general applicability for the investigation of biomolecular events with single-molecule resolution in defined nanoarrays configurations and with orientational control of the (bio)molecule of interest.