Department of Chemistry and Biochemistry and Biodesign Institute, Arizona State University, Tempe, AZ, USA.
Photochem Photobiol. 2012 Jul-Aug;88(4):782-91. doi: 10.1111/j.1751-1097.2012.01121.x. Epub 2012 Mar 28.
We have investigated the association interactions between the fluorescent dyes TAMRA, Cy3B and Alexa-546 and the DNA deoxynucleoside monophosphates by means of fluorescence quenching and fluorescence correlation spectroscopy (FCS). The interactions of Cy3B and TAMRA with the nucleotides produce a decrease in the apparent diffusion coefficient of the dyes, which result in a shift toward longer times in the FCS autocorrelation decays. Our results with Cy3B demonstrate the existence of Cy3B-nucleotide interactions that do not affect the fluorescence intensity or lifetime of the dye significantly. The same is true for TAMRA in the presence of dAMP, dCMP and dTMP. In contrast, the diffusion coefficient of Alexa 546 remains practically unchanged even at high concentrations of nucleotide. These results demonstrate that interactions between this dye and the four dNMPs are not significant. The presence of the negatively charged sulfonates and the bulky chlorine atoms in the phenyl group of Alexa 546 possibly prevent strong interactions that are otherwise possible for TAMRA. The characterization of dye-DNA interactions is important in biophysical research because they play an important role in the interpretation of energy transfer experiments, and because they can potentially affect the structure and dynamics of the DNA.
我们通过荧光猝灭和荧光相关光谱（FCS）研究了荧光染料 TAMRA、Cy3B 和 Alexa-546 与脱氧核苷酸单磷酸之间的缔合相互作用。Cy3B 和 TAMRA 与核苷酸的相互作用导致染料的表观扩散系数降低，从而导致 FCS 自相关衰减向更长时间移动。我们用 Cy3B 得到的结果表明，Cy3B-核苷酸相互作用的存在不会显著影响染料的荧光强度或寿命。在 dAMP、dCMP 和 dTMP 存在的情况下，TAMRA 也是如此。相比之下，即使在核苷酸的高浓度下，Alexa 546 的扩散系数仍保持基本不变。这些结果表明，该染料与四种 dNMP 之间的相互作用并不显著。Alexa 546 苯环上的磺酸基和大氯原子带负电荷，可能阻止了与 TAMRA 可能发生的强相互作用。染料-DNA 相互作用的特性在生物物理研究中很重要，因为它们在解释能量转移实验中起着重要作用，并且它们可能会影响 DNA 的结构和动态。
