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An ideal nanofabrication method should allow the organization ofnanoparticles and molecules with nanometric positional precision,stoichiometric control and well-defined orientation. The DNA origami techniquehas evolved into a highly versatile bottom-up nanofabrication methodology thatfulfils almost all of these features. It enables the nanometric positioning ofmolecules and nanoparticles with stoichiometric control, and even theorientation of asymmetrical nanoparticles along predefined directions. However,orienting individual molecules has been a standing challenge, mainly due tounspecific electrostatic interactions. Here, we show how single molecules,namely Cy5 and Cy3 fluorophores, can be incorporated in a DNA origami withcontrolled orientation by doubly linking them to oligonucleotide strands thatare hybridized while leaving enough unpaired bases to induce a stretchingforce. Particularly, we explore the effects of leaving 0, 2, 4, 6, and 8unpaired bases and find extreme orientations for 0 and 8 unpaired bases,corresponding to the molecules being perpendicular and parallel to the DNAdouble helix, respectively. We foresee that these results will expand theapplication field of DNA origami towards the fabrication of nanodevicesinvolving a wide range of orientation-dependent molecular interactions, such asenergy transfer, intermolecular electron transport, catalysis, excitondelocalization, or the electromagnetic coupling of a molecule to specificresonant nano-antennas modes.

DNA self-assembly of single molecules with deterministic position and orientation