Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.
Chemistry. 2011 Sep 5;17(37):10388-96. doi: 10.1002/chem.201100215. Epub 2011 Aug 4.
Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90% in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis.
T4 DNA 连接酶在经修饰的 DNA 模板上进行高效的 DNA 缺口密封，该模板中引入了诸如偶氮苯的嵌入剂。嵌入剂通过插入 DNA 主链的 D-苏糖醇接头连接。尽管连接点处的模板结构与天然 DNA 完全不同，但在大多数情况下，两种寡核苷酸可以以高于 90%的产率连接。对序列依赖性的系统研究表明，连接效率随与偶氮苯部分相邻的碱基对而有很大差异。有趣的是，当引入的偶氮苯在经受 UV 光（320-380nm）照射时异构化为顺式时，所有研究的序列的连接速度都大大加快。这些意外的连接可能为在由于引入的功能分子阻止 DNA 合成而无法直接进行 PCR 的情况下将功能分子引入长 DNA 链提供了一种新方法。还基于动力学分析讨论了这种意外酶促作用的生物学意义。
