Seeman N C, Chen J H, Kallenbach N R
Department of Chemistry, New York University, New York 10003.
Electrophoresis. 1989 May-Jun;10(5-6):345-54. doi: 10.1002/elps.1150100512.
Gel electrophoresis has provided much of the detailed information we have about the properties of DNA junctions, stable branched molecules formed from oligonucleotide or polynucleotide strands. Here we review these applications, and present the results of an electrophoretic investigation of conformationally restricted junctions formed by covalently connecting two different pairs of strands in a junction with four arms. Native gel electrophoresis is employed to establish the formation and stoichiometry of the multistrand complexes. Ferguson analysis of native gel mobility shows that junctions have retardation coefficients that are distinct from those of linear DNA duplexes. Denaturing gel electrophoresis is the primary tool for characterizing junctions that have been covalently linked together to form both linear and macrocyclic oligomers of junctions (oligojunctions). Radioactively labelled strands enable one to monitor the progress of the ligation reaction: both linear and closed cyclic molecules result, and these can be distinguished by applying Ferguson analysis to denaturing gels. Combinations of exonuclease III, restriction enzymes and sequencing reactions have been applied to oligojunction molecules, and the results are all analyzed on denaturing gels. Junctions containing intramolecular "tethers" that restrict the conformation freedom of the complex comprise a new system for analyzing the conformations of branched molecules. In these tethered junctions, the ability of arms to move relative to each other is restricted substantially by covalently connecting pairs of arms in the original complex with short, flexible loops. The two tethers used here constrain the helical domains of the structure to be roughly parallel or anti-parallel. In this article, we use Ferguson analysis to compare two tethered junctions with an untethered junction. At high gel concentrations, the mobility of the untethered complex is found to be closer to that of the molecule tethered anti-parallel than to the one tethered parallel. Curvature in the Ferguson plots for all three of these junctions is detected over a range of compositions. At low gel concentrations, differences in electrophoretic mobility persist, suggesting that the untethered junction differs in charge as well as conformational freedom from the tethered analogs. We expect that studies of this kind will be able to define the conformational repertoire of junctions of different kinds, and to explore the effects of electrophoresis on these states.
凝胶电泳为我们提供了许多关于DNA连接体特性的详细信息，DNA连接体是由寡核苷酸或多核苷酸链形成的稳定分支分子。在此，我们回顾这些应用，并展示对由四条臂的连接体中两对不同链共价连接形成的构象受限连接体进行电泳研究的结果。采用非变性凝胶电泳来确定多链复合物的形成和化学计量。对非变性凝胶迁移率进行的弗格森分析表明，连接体的阻滞系数与线性DNA双链体的不同。变性凝胶电泳是表征已共价连接在一起形成连接体的线性和大环低聚物（寡连接体）的主要工具。放射性标记的链使人们能够监测连接反应的进程：线性和闭环分子都会产生，通过对变性凝胶应用弗格森分析可以区分它们。外切核酸酶III、限制性内切酶和测序反应的组合已应用于寡连接体分子，结果均在变性凝胶上进行分析。含有限制复合物构象自由度的分子内“系链”的连接体构成了一个用于分析分支分子构象的新系统。在这些系链连接体中，臂相对于彼此移动的能力通过用短的柔性环共价连接原始复合物中的臂对而受到显著限制。这里使用的两条系链将结构的螺旋结构域限制为大致平行或反平行。在本文中，我们使用弗格森分析来比较两个系链连接体与一个非系链连接体。在高凝胶浓度下，发现非系链复合物的迁移率更接近反平行系链分子而不是平行系链分子。在一系列组成范围内检测到这三种连接体的弗格森图中都有曲率。在低凝胶浓度下，电泳迁移率的差异仍然存在，这表明非系链连接体在电荷以及构象自由度方面与系链类似物不同。我们预计这类研究将能够定义不同类型连接体的构象库，并探索电泳对这些状态的影响。
