生命金属寡核苷酸螯合物的制备及反应机理

CHELATES OF BIOMETAL IONS WITH EDTA COVALENTLY LINKED TO OLIGONUCLEOTIDES:PREPARATION AND THEIR CLEAVAGE MECHANISM

制备了５′端共价连接ＥＤＴＡ的寡聚核苷酸与生命金属的螯合物ＯＤＮ－５′－ＥＤＴＡＭ（ｎ＋），其中Ｍ（ｎ＋）为Ｆｅ（Ⅱ），Ｃｏ（Ⅱ）或Ｃｕ（Ⅱ）；分析、计算了这３个螯合物形成的最佳ｐＨ值范围，ＯＤＮ－ＥＤＴＡＦｅ（Ⅱ）为ｐＨ５．８～８．６，ＯＤＮ－ＥＤＴＡＣｏ（Ⅱ）为ＰＨ４．６～８．１，ＯＤＮ－ＥＤＴＡＣｕ（Ⅱ）为ｐＨ３．４～５．７；在此条件下，剪切反应必须的Ｍｇ２＋并不与Ｆｅ（Ⅱ），Ｃｏ（Ⅱ）或Ｃｕ（Ⅱ）竞争ＥＤＴＡ，也不会形成沉淀；讨论了ＯＤＮ－ＥＤＴＡＭ（ｎ＋）切割ＤＮＡ双链的反应机理：修饰寡核苷酸通过氢键在ＤＮＡ双链的大沟结合形成三链体，在Ｏ２和还原剂ＤＴＴ作用下，Ｆｅ作为催化剂，产生羟基自由基，氧化糖环，切割ＥＤＴＡＦｅ（Ⅱ）附近的ＤＮＡ双链．

The chelates of biometal ions with EDTA covalently linked to the 5′-end of oligodeoxynucleotides(ODN), i. e. , ODN-5′-EDTA·M(Ⅱ), are prepared, in which M(Ⅱ)is Fe(Ⅱ), Co(Ⅱ)or Cu(Ⅱ). The optimization of pH value for forming these chelates is calculated. For ODN-5′-EDTA·Fe(Ⅱ) pH value is 5 8 to 8 6, pH 4 6～8 1 for ODN-5′-EDTA·Co(Ⅱ), pH 3. 4～5. 7 for ODN-5′-EDTA·Cu(Ⅱ). Under such pH value conditions neither can Mg 2+ ions, necessary for cleavage reaction, be competitive with Fe(Ⅱ), Co(Ⅱ) or Cu(Ⅱ) to form EDTA Chelate, nor can it be precipitated. The cleavage mechanism of ODN-5′-EDTA·Fe(Ⅱ) for DNA duplex is discussed. Modified ODN binds with DNA duplex in the major groove via hydrogen bond to form triple helix . In the presence of oxygen and reducing agent DTT(dithiothreitol) hydroxyl radicals species are generated as intermediates by the catalysis of metal iron, and then oxidize the ribo ring and cut the double stranded DNA at the sites close to EDTA·M(Ⅱ).