Protozanova E, Macgregor R B
Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, ON, Canada.
Electrophoresis. 1999 Jul;20(10):1950-7. doi: 10.1002/(SICI)1522-2683(19990701)20:10<1950::AID-ELPS1950>3.0.CO;2-V.
DNA frayed wires are extremely stable multistranded complexes arising from the association of oligonucleotides with long terminal runs of consecutive guanines. Frayed wires originating from d(A15G15) have multiple binding sites for short complementary oligonucleotides such as dT10. We examine unusual band patterns obtained when complexes formed between dT10 and DNA frayed wires are resolved on nondenaturing polyacrylamide gels. Since the lifetime of the dT10-frayed wire complexes is shorter than the time of the gel run, the interaction between the components during the gel electrophoresis affects their band patterns. We have conducted chasing experiments to show that (i) the binding of dT10 to the frayed wires can occur during gel electrophoresis, and (ii) dissociation of the complexes occurs during the gel run. Rapid repetitive dissociation-reassociation of the complexes leads to a constant partitioning of dT10 between their binding sites within frayed wires. Consequently, complexes composed of frayed wires and various numbers of bound ligands appear on the gel as a single well-defined band. The mobilities of these bands decrease continuously with the concentration of the ligand reaching saturation when all the binding sites are occupied. This characteristic pattern is observed only for relatively unstable interactions. Longer ligands, i.e., oligonucleotides with higher affinity towards the binding sites, cease to exhibit the dynamic character of interaction during gel electrophoresis. These ligands form long-lived complexes with the frayed wires that appear on the gel as faint smeared bands reflecting the presence of multiple stable complexes.
DNA 磨损链是由寡核苷酸与连续鸟嘌呤的长末端序列结合形成的极其稳定的多链复合物。源自 d(A15G15) 的磨损链对短互补寡核苷酸（如 dT10）具有多个结合位点。我们研究了在非变性聚丙烯酰胺凝胶上解析 dT10 与 DNA 磨损链形成的复合物时获得的异常条带模式。由于 dT10 - 磨损链复合物的寿命短于凝胶电泳运行时间，凝胶电泳过程中各组分之间的相互作用会影响它们的条带模式。我们进行了追踪实验以表明：（i）dT10 与磨损链的结合可在凝胶电泳过程中发生，以及（ii）复合物的解离在凝胶电泳运行过程中发生。复合物的快速重复解离 - 重新结合导致 dT10 在磨损链内的结合位点之间持续分配。因此，由磨损链和各种数量的结合配体组成的复合物在凝胶上表现为单一清晰定义的条带。当所有结合位点被占据时，这些条带的迁移率随着配体浓度的增加而持续降低直至饱和。这种特征模式仅在相对不稳定的相互作用中观察到。较长的配体，即对结合位点具有更高亲和力的寡核苷酸，在凝胶电泳过程中不再表现出相互作用的动态特征。这些配体与磨损链形成长寿命复合物，在凝胶上表现为模糊的拖带，反映了多种稳定复合物的存在。
