In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5'-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive Förster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye-DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye-DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680-dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor-acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large Förster distance of ~7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins.
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