An
ideal nanofabrication method should allow the organization of
nanoparticles and molecules with nanometric positional precision,
stoichiometric control, and well-defined orientation. The DNA origami
technique has evolved into a highly versatile bottom-up nanofabrication
methodology that fulfils almost all of these features. It enables
the nanometric positioning of molecules and nanoparticles with stoichiometric
control, and even the orientation of asymmetrical nanoparticles along
predefined directions. However, orienting individual molecules has
been a standing challenge. Here, we show how single molecules, namely,
Cy5 and Cy3 fluorophores, can be incorporated in a DNA origami with
controlled orientation by doubly linking them to oligonucleotide strands
that are hybridized while leaving unpaired bases in the scaffold.
Increasing the number of bases unpaired induces a stretching of the
fluorophore linkers, reducing its mobility freedom, and leaves more
space for the fluorophore to accommodate and find different sites
for interaction with the DNA. Particularly, we explore the effects
of leaving 0, 2, 4, 6, and 8 bases unpaired and find extreme orientations
for 0 and 8 unpaired bases, corresponding to the molecules being perpendicular
and parallel to the DNA double-helix, respectively. We foresee that
these results will expand the application field of DNA origami toward
the fabrication of nanodevices involving a wide range of orientation-dependent
molecular interactions, such as energy transfer, intermolecular electron
transport, catalysis, exciton delocalization, or the electromagnetic
coupling of a molecule to specific resonant nanoantenna modes.