Chen Wen, Kitamura Yoshihito, Zhou Jing-Min, Sumaoka Jun, Komiyama Makoto
Contribution from the Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
J Am Chem Soc. 2004 Aug 25;126(33):10285-91. doi: 10.1021/ja048953a.
By using two oligonucleotide additives that bear a monophosphate group at the termini through various linkers, gap structures were formed at predetermined positions in substrate DNA, and the monophosphate groups were placed at both edges of these gaps. At pH 7.0 and 37 degrees C, the phosphodiester linkages in the gap sites were efficiently and selectively hydrolyzed by Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). The linkages in the middle of the gaps were predominantly hydrolyzed. Compared with DNA scission using oligonucleotide additives that bear no terminal monophosphate, the present scission was much faster (22-fold for a 3-base gap and 14-fold for a 5-base gap) and more site selective. Introduction of one monophosphate group to either edge of the gaps was also effective for promotion of both site selectivity and scission rate. The monophosphate group(s) at the gap site recruits the Ce(IV) to the target site and magnifies the difference in intrinsic reactivity between the target site and the others. Even at higher reaction temperatures, the site selectivity remained satisfactorily high. Furthermore, the fragments formed by the site-selective scission were connected with various oligonucleotides by using DNA ligase, producing desired recombinant DNAs.
通过使用两种在末端通过各种连接子带有单磷酸基团的寡核苷酸添加剂，在底物DNA的预定位置形成了缺口结构，并且单磷酸基团位于这些缺口的两侧。在pH 7.0和37℃下，Ce(IV)/EDTA络合物（EDTA = 乙二胺-N,N,N',N'-四乙酸）能有效且选择性地水解缺口中的磷酸二酯键。缺口中间的键主要被水解。与使用末端不带有单磷酸的寡核苷酸添加剂进行DNA切割相比，当前的切割速度要快得多（对于3个碱基的缺口快22倍，对于5个碱基的缺口快14倍）且位点选择性更高。在缺口的任一边引入一个单磷酸基团对于提高位点选择性和切割速率也很有效。缺口中的单磷酸基团将Ce(IV)募集到靶位点，并放大了靶位点与其他位点之间固有反应性的差异。即使在较高的反应温度下，位点选择性仍保持令人满意的高水平。此外，通过位点选择性切割形成的片段通过使用DNA连接酶与各种寡核苷酸连接，从而产生所需的重组DNA。
