Kinetic consequences of covalent linkage of DNA binding polyamides
Kinetic consequences of covalent linkage of DNA binding polyamides
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DOI：
10.1021/bi0022339
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发表时间：
2001-01-09
期刊：
影响因子：
2.9
通讯作者：
Crothers, DM
中科院分区：
文献类型：
--
作者：
Baliga, R;Baird, EE;Crothers, DM
由N-甲基吡咯（Py）和N-甲基咪唑（Im）亚基组成的聚酰胺可以以亚纳摩尔的亲和力和高特异性结合在DNA的小沟中的预定序列处。使用γ-氨基丁酸接头的聚合物亚基的共价连接已显示出增加聚酰胺的亲和力和特异性。使用荧光检测停流分析，我们已经研究了一系列聚酰胺代表未连接的，发夹和环状类似物的四环聚酰胺ImPyPyPy-beta -Dp的缔合和解离动力学的差异。而在未连接和共价连接的聚酰胺之间的平衡缔合常数中看到的大的差异主要是由于较高的缔合速率常数，每个聚酰胺之间的匹配和错配位点的歧视可以归因于在很大程度上在其解离速率常数的差异。这种动力学行为的后果为未来的设计进行了讨论。
Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) subunits can bind in the minor groove of DNA at predetermined sequences with subnanomolar affinity and high specificity. Covalent linkage of polymer subunits using a gamma -aminobutyric acid linker has been shown to increase both the affinity and specificity of polyamides. Using a fluorescence detected stopped-flow assay, we have studied the differences in association and dissociation kinetics of a series of polyamides representing unlinked, hairpin and cyclic analogues of the four ring polyamide ImPyPyPy-beta -Dp. Whereas the large differences seen in the equilibrium association constants between the unlinked and covalently linked polyamides are primarily due to higher association rate constants, discrimination between matched and mismatched sites by each polyamide can be ascribed in large part to differences in their dissociation rate constants. The consequences of this kinetic behavior for future design are discussed.