Sequence-Directed Control of Cyanine Dye Stacking Geometry within DNA Duplexes

The photophysical properties of dye aggregates are governed by their supramolecular stacking geometries, such as H- and J-type arrangements. Structural DNA nanotechnology provides a programmable platform for the spatially precise organization of chromophores, yet how nearest-neighbor base-pair sequences specifically regulate dye-stacking modes remains unclear. Here, we demonstrated that the nearest-neighbor base-pair sequence within a DNA duplex functions as a sequence-encoded regulator that directs the stacking geometry of covalently tethered cyanine-dye dimers. By systematically varying the local base-pair environment adjacent to internally incorporated Cy5 and Cy3 dyes, we show that C/G neighbors bias Cy5 dimers toward H-type cofacial stacking, whereas A/T base pairs promote J-type head-to-tail arrangements, and C/T neighboring sequences promote an oblique stacking geometry. In contrast, Cy3 dimers predominantly adopt J-type configurations irrespective of sequence context, revealing a pronounced dye-dependent selectivity in how local sequence controls stacking. These findings indicate that DNA can function not only as a passive scaffold but also as an active, sequence-dependent microenvironment that modulates supramolecular order through nearest-neighbor variation, providing a minimal and predictive strategy for engineering excitonic nanomaterials with tunable optical and chiroptical properties.