Oxazine Dye-Conjugated DNA Oligonucleotides: Forster Resonance Energy Transfer in View of Molecular Dye-DNA Interactions
Oxazine Dye-Conjugated DNA Oligonucleotides: Forster Resonance Energy Transfer in View of Molecular Dye-DNA Interactions
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DOI：
10.1021/bc200379y
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发表时间：
2011-12-01
影响因子：
4.7
通讯作者：
Kumke, Michael U.
中科院分区：
文献类型：
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作者：
Kupstat, Annette;Ritschel, Thomas;Kumke, Michael U.
本工作研究了两种恶嗪染料(ATTO 610和ATTO 680)通过C6-氨基连接到不同长度的短单链和双链DNA(分别为单链和双链DNA)5‘端的光物理性质。这两种恶嗪染料之所以被选中，是因为它们具有良好的光谱重叠、高消光系数和ATTO 610的高荧光量子产率，使它们成为具有吸引力的Forster共振能量转移(FRET)对，用于远红外光谱范围的生物分析。为了确定可能导致光物理变化的分子染料-DNA相互作用，我们进行了详细的光谱研究，包括时间分辨荧光各向异性和荧光相关光谱测量。由于DNA偶联的影响，两种染料的吸收峰和荧光峰都发生了红移，荧光衰减时间增加。此外，共轭ATTO 610的吸收光谱被展宽，并观察到双重荧光发射。这两种染料都与ssDNA和dsDNA发生了空间相互作用。从ssDNA到dsDNA偶联物，染料与DNA的相互作用增强，指向与特定dsDNA结构域(如双螺旋的顶部)的相互作用。尽管这些相互作用部分地阻止了染料-连接物的旋转，但至少一种染料的自由(不受阻碍的)旋转迁移率促进了双标记ATTO 610/ATTO 680-dsDNA偶联物中转变偶极矩的适当排列，从而实现了FRET的成功。考虑到用于确定给体-受体距离的连接基的高度灵活性，FRET的理论参数和实验参数得到了很好的一致。相当大的Forster距离类似于7 nm，建议使用这种FRET对不仅用于检测活细胞中核酸之间的结合反应，而且还用于监测较大生物分子(如蛋白质)的相互作用。
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 Forster 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 Forster distance of similar to 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.