Kumke M U, Li G, McGown L B, Walker G T, Linn C P
P. M. Gross Chemical Laboratory, Department of Chemistry, Duke University, Durham, North Carolina 27708-0346, USA.
Anal Chem. 1995 Nov 1;67(21):3945-51. doi: 10.1021/ac00117a020.
Fundamental aspects of the application of fluorescence anisotropy to detect the hybridization of fluorescein-labeled DNA oligomers were explored. The oligomers included a binding site for the EcoRI restriction enzyme, which binds to double-stranded DNA and is used in this work to enhance the difference between the anisotropies of the single-stranded and double-stranded oligomers by increasing the effective volume of the latter. The fluorescence anisotropy increases upon hybridization and further upon binding of EcoRI to the double strand. By varying the length of the tether used to attach the fluorescein to the 5' end of the oligonucleotide, it was found that a 6-carbon tether was optimal, providing the most dramatic increases in anisotropy in the presence of EcoRI. Dynamic fluorescence anisotropy (DFA) provided insight into the increases in steady-state anisotropy. In most cases, the best fits to the DFA data were obtained using a biexponential decay model, which describes an anisotropic rotator. Upon hybridization, the faster rotational motion is more hindered, and the contribution of the slower rotational component is increased. This effect is enhanced by binding of EcoRI to the double strand, especially when the EcoRI binding site is near the fluorescein at the 5' end and the tether length is in the optimal range. Because the rotational correlation time of the slower anisotropy decay component is much longer than the fluorescence lifetime, it is possible in some cases to reduce the anisotropic rotator model to the special limiting case of a hindered rotator.
探索了应用荧光各向异性检测荧光素标记的DNA寡聚物杂交的基本方面。这些寡聚物包含EcoRI限制酶的结合位点，该酶与双链DNA结合，在本研究中用于通过增加双链寡聚物的有效体积来增强单链和双链寡聚物各向异性之间的差异。杂交时荧光各向异性增加，EcoRI与双链结合时进一步增加。通过改变用于将荧光素连接到寡核苷酸5'端的连接链长度，发现6碳连接链是最佳的，在存在EcoRI的情况下各向异性增加最为显著。动态荧光各向异性（DFA）为稳态各向异性的增加提供了深入了解。在大多数情况下，使用双指数衰减模型能最好地拟合DFA数据，该模型描述了一个各向异性旋转体。杂交时，较快的旋转运动受到更大阻碍，较慢旋转成分的贡献增加。EcoRI与双链结合会增强这种效应，特别是当EcoRI结合位点靠近5'端的荧光素且连接链长度处于最佳范围时。由于较慢各向异性衰减成分的旋转相关时间远长于荧光寿命，在某些情况下有可能将各向异性旋转体模型简化为受阻旋转体的特殊极限情况。
