Laib Stephan, Seeger Stefan
Institute for Physical Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
J Fluoresc. 2004 Mar;14(2):187-91. doi: 10.1023/b:jofl.0000016290.34070.ee.
Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to determine distances between chromophores bound to macromolecules, since the efficiency of the energy transfer from an initially excited donor to an acceptor strongly depends on the distance between the two dye molecules. The structure of the noncovalent complex of double-strand DNA (dsDNA) with thiazol orange dimers (TOTO) allows FRET analysis of two intercalated chromophores. By intercalation of two different TOTO dyes we observe an energy transfer from TOTO-1 as donor and TOTO-3 as acceptor. In this manner we are able to determine the mean distance between two proximate TOTO molecules bound to dsDNA. Thus the maximum number of binding positions for this type of intercalation dyes in the dsDNA can be obtained. Furthermore the dependency of the acceptor emission on the donor concentration is analysed. The emission of TOTO-3 reaches a maximum when the acceptor-to-donor ratio is 1:10.
荧光共振能量转移(FRET)是一种用于确定与大分子结合的发色团之间距离的强大工具,因为从初始激发的供体到受体的能量转移效率强烈依赖于两个染料分子之间的距离。双链DNA(dsDNA)与噻唑橙二聚体(TOTO)的非共价复合物结构允许对两个插入的发色团进行FRET分析。通过插入两种不同的TOTO染料,我们观察到从作为供体的TOTO-1到作为受体的TOTO-3的能量转移。通过这种方式,我们能够确定与dsDNA结合的两个相邻TOTO分子之间的平均距离。因此,可以获得这种类型的插入染料在dsDNA中的最大结合位置数。此外,还分析了受体发射对供体浓度的依赖性。当受体与供体的比例为1:10时,TOTO-3的发射达到最大值。