1 research outputs found
Oxazine Dye-Conjugated DNA Oligonucleotides: Förster Resonance Energy Transfer in View of Molecular Dye–DNA Interactions
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