Site-selective DNA hydrolysis by combining Ce(IV)/EDTA with monophosphate-bearing oligonucleotides and enzymatic ligation of the scission fragments
Site-selective DNA hydrolysis by combining Ce(IV)/EDTA with monophosphate-bearing oligonucleotides and enzymatic ligation of the scission fragments
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DOI：
10.1021/ja048953a
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发表时间：
2004-08-25
影响因子：
15
通讯作者：
Komiyama, M
中科院分区：
文献类型：
--
作者：
Chen, W;Kitamura, Y;Komiyama, M
通过使用两个末端带有单磷酸基团的寡核苷酸添加剂，通过不同的连接子，在底物DNA的预定位置形成间隙结构，并将单磷酸基团放置在这些间隙的两侧。在pH 7.0和37℃时，Ce(IV)/EDTA络合物(EDTA=乙二胺-N，N，N‘，N’-四乙酸乙酯)能有效、选择性地催化裂解位上的磷酸二酯键。缝隙中间的连接主要是被水解的。与使用不含末端一磷酸的寡核苷酸添加剂进行DNA切割相比，目前的切割速度要快得多(3碱基缺口是22倍，5碱基缺口是14倍)，而且更具位点选择性。在空隙的两边引入一个单磷酸基团也有效地提高了位置选择性和断裂率。间隙位上的单磷酸基团(S)将Ce(IV)招募到靶位，放大了靶位与其他位之间的本征反应性差异。即使在较高的反应温度下，位置选择性仍然令人满意地保持高。此外，通过DNA连接酶将定点切割形成的片段与不同的寡核苷酸连接，得到所需的重组DNA。
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 degreesC, 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.