Metelev Valeri, Zhang Surong, Tabatadze David, Kumar Anand T N, Bogdanov Alexei
The Laboratory of Molecular Imaging Probes, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Mol Biosyst. 2013 Oct;9(10):2447-53. doi: 10.1039/c3mb70108e.
We report a general phenomenon of the formation of either a fluorescent or an entirely quenched oligodeoxynucleotide (ODN) duplex system by hybridizing pairs of complementary ODNs with identical chemical composition. The ODNs carried internucleoside tether-linked cyanines, where the cyanines were chosen to form a Förster's resonance energy transfer (FRET) donor-acceptor pair. The fluorescent and quenched ODN duplex systems differed only in that the cyanines linked to the respective ODNs were linked either closer to the 5'- or 3'-ends of the molecule. In either case, however, the dyes were separated by an identical number (7 or 8) of base pairs. Characterization by molecular modeling and energy minimization using a conformational search algorithm in a molecular operating environment (MOE) revealed that linking of the dyes closer to the 5'-ends resulted in their reciprocal orientation across the major groove which allowed a closely interacting dye pair to be formed. This overlap between the donor and acceptor dye molecules resulted in changes in absorbance spectra consistent with the formation of H-aggregates. Conversely, dyes linked closer to 3'-ends exhibited emissive FRET and formed a pair of dyes that interacted with the DNA helix only weakly. Induced CD spectra analysis suggested that interaction with the double helix was weaker than in the case of the closely interacting cyanine dye pair. Linking the dyes such that the base pair separation was 10 or 0 favored energy transfer with subsequent acceptor emission. Our results suggest that when interpreting FRET measurements from nucleic acids, the use of a "spectroscopic ruler" principle which takes into account the 3D helical context of the double helix will allow more accurate interpretation of fluorescence emission.
我们报道了一种普遍现象，即通过将具有相同化学组成的互补寡脱氧核苷酸（ODN）对进行杂交，形成荧光或完全淬灭的寡脱氧核苷酸双链体系统。这些ODN带有核苷间连接的花青染料，其中选择的花青染料形成弗斯特共振能量转移（FRET）供体-受体对。荧光和淬灭的ODN双链体系统的区别仅在于，连接到各自ODN上的花青染料与分子的5'-或3'-末端连接得更近。然而，在任何一种情况下，染料之间都被相同数量（7或8）的碱基对隔开。通过在分子操作环境（MOE）中使用构象搜索算法进行分子建模和能量最小化来表征，结果表明，将染料连接到更靠近5'-末端会导致它们在大沟中相互反向排列，从而形成紧密相互作用的染料对。供体和受体染料分子之间的这种重叠导致吸收光谱的变化，这与H-聚集体的形成一致。相反，连接到更靠近3'-末端的染料表现出发射性FRET，并形成一对与DNA螺旋相互作用较弱的染料。诱导圆二色光谱分析表明，与双螺旋的相互作用比紧密相互作用的花青染料对的情况弱。将染料连接成碱基对间隔为10或0有利于能量转移并随后产生受体发射。我们的结果表明，在解释来自核酸的FRET测量时，使用考虑双螺旋三维螺旋背景的“光谱尺”原理将允许对荧光发射进行更准确的解释。
