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

Abstract

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.

Accelerated single photon emission from dye molecule-driven nanoantennas assembled on DNA

“…As mentioned earlier, cationic dyes tend to bind to DNA, hindering their rotational degrees of freedom 23 . In practice, large dye molecules typically interact with the DNA grooves, favouring a π/4 molecular orientation 27 .…”

“…Single lifetimes and stable fluorescence time traces indicate that the SM orientations are stable during the measurement. Complete rotational hindrance of similar cationic dye molecules bound to double-stranded DNA was recently analysed in time-resolved anisotropy measurements 23 . Figure 2 depicts the distributions of measured normalized decay rate enhancements Γ/〈Γ 0 〉, with 〈Γ 0 〉 the average decay rate of isolated ATTO647N molecules.…”

Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5′‐terminal Cy5 dyes

“…However, their susceptibility to photobleaching, the absence of fluorescence at low pH and their broad emission spectra have favoured the development of novel fluorescent dyes. Cyanines dyes (Cy dyes) , BODIPY dyes , Alexa dyes and ATTO dyes are versatile groups of fluorescent dyes with greater sensitivity, stability and lower background interferences. Table lists the spectroscopic and physicochemical features of these fluorescent dyes.…”

Photonic Engineering of Hybrid Metal–Organic Chromophores

“…To further confirm that our studied samples correspond to purified suspensions of AuNP monomers and dimers linked to one ATTO647N-modified DNA strand, we compare experimental luminescence lifetimes with theoretical estimates of t. In practice, fluorescence lifetimes are estimated by computing the power dissipated by a point dipole in AuNP monomers and dimers using classical electromagnetic theory. [28,29] Since dye-modified DNA strands feature fixed [30] but uncontrolled molecular orientations, we take an isotropic distribution of the angle between the molecular transition dipole and the particles. The emitter-AuNP distances are set at 6.5 and 9.5 nm for the 30 bp and 50 bp linkers, in good agreement with cryo-EM and scattering spectroscopy.…”