Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
Bioconjug Chem. 2011 Aug 17;22(8):1491-502. doi: 10.1021/bc100485f. Epub 2011 Jul 22.
We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.
我们合成了具有多个噻唑橙（TO）嵌入染料的荧光 DNA 双链体，这些染料通过三唑键共价连接到 DNA 上。嵌入染料稳定了双链体，使其不易热变性，并在光谱的绿色区域显示出明亮的荧光。通过添加共价连接到 DNA 双链体上的能量接受 Cy3 或 Cy5 染料，可以将发射颜色变为橙色或红色。然后，将染料修饰的 DNA 双链体连接到第二抗体上，用于在果蝇胚胎中进行中心体的细胞内荧光成像。在供体（TO）和受体（Cy5）通道中都观察到明亮的荧光焦点，因为能量转移效率适中。监测 Cy5 发射通道显著降低了背景信号，因为能量转移允许发射波长的大幅移动。
