ORIENTATION OF LARGE DNA DURING FREE SOLUTION ELECTROPHORESIS STUDIED BY LINEAR DICHROISM
ORIENTATION OF LARGE DNA DURING FREE SOLUTION ELECTROPHORESIS STUDIED BY LINEAR DICHROISM
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DOI：
10.1039/ft9938902791
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发表时间：
1993-08-07
期刊：
影响因子：
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通讯作者：
NORDEN, B
中科院分区：
文献类型：
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作者：
JONSSON, M;JACOBSSON, U;NORDEN, B
使用相位调制检测线性二色性，在游离溶液（25 mmol dm-3 离子强度）中的电泳条件下研究了 DNA T7（40 kbp（双匕首））、T5（120 kbp）和 T2（170 kbp）的链取向和无场衰减。对于较大的 T5 和 T2 DNA，稳态取向不显示克尔定律行为，但随场强大致线性增加，取向因子在 100 V cm-1 时约为 3 x 10(-3)。当受到电场时，DNA 在 0.1-5 秒内显示出方向超调，该超调随着 DNA 大小的增加而强烈增加，并且场强越高，出现的速度越快。当场关闭时，DNA 方向的主要部分在 10 毫秒内松弛，而较小部分则衰减得更慢：T2 为 0.3 秒，与 Zimm-Rouse 链的最慢松弛相当。观察到的定向现象对于 DNA 自由溶液毛细管电泳可能很重要。 DNA 排列机制的问题（偶极子取向还是电泳取向？）在现有的链状大分子理论中无法得到充分解决。在等待模拟结果时，推测大 DNA 的过冲现象和缓慢衰减成分可能代表 DNA 线圈的重复变形，类似于最近在凝胶电泳期间观察到的大 DNA 的变形。快速场反转不会导致纯 DNA 的稳态方向发生任何下降；然而，在与重组酶蛋白 RecA 形成的复合物中，RecA 显然以极性方式堆积在 DNA 外部，形成一个大的永久偶极子，明显的倾角表明粒子在场反转时发生 180 度旋转。电光池的设计将电渗透和对流的影响降至最低。 T2 和 T7 DNA（地面）的实验在探空火箭飞行期间的微重力条件下进行了复制，方向动力学没有任何明显差异。
DNA T7 (40 kbp(double dagger)), T5 (120 kbp) and T2 (170 kbp) have been studied under electrophoretic conditions in free solution (25 mmol dm-3 ionic strength) regarding chain orientation and field-free decay using phase-modulation detected linear dichroism. The steady-state orientation does not show Kerr-law behaviour for the larger T5 and T2 DNAs but increases roughly linearly with field strength, the orientation factor being around 3 x 10(-3) at 100 V cm-1. When subjected to the electric field DNA displays within 0.1-5 s an orientation overshoot which increases strongly with DNA size and comes faster the higher the field strength. When the field is switched off a major portion of the DNA orientation has relaxed within 10 ms while a smaller portion decays more slowly: 0.3 s for T2, comparable with the slowest relaxation of a Zimm-Rouse chain. The observed orientation phenomena could be important in free-solution capillary electrophoresis of DNA. The question of the mechanism by which the DNA is aligned (dipole or electrophoretic orientation?) cannot be addressed adequately within existing theories for chain-like macromolecules. Awaiting results from simulations it is speculated that the overshoot phenomenon and the slow decay component of large DNA may represent reptative deformations of the DNA coil similar to those recently observed for large DNA during gel electrophoresis. Rapid field reversal did not lead to any dip in steady-state orientation for the pure DNA; however, in a complex with the recombinase protein RecA which stacks outside DNA, apparently in a polar way to form a large permanent dipole, a pronounced dip suggests that there is a 180-degrees rotation of the particle upon field reversal. The electro-optical cell was designed to give minimal influence from electro-osmosis and convection. The experiments on T2 and T7 DNA (on the ground) were replicated under microgravity conditions during a sounding-rocket flight without any noticeable differences in orientation dynamics.