Design of a synthetic nuclease: DNA hydrolysis by a zinc-binding peptide tethered to a rhodium intercalator
Design of a synthetic nuclease: DNA hydrolysis by a zinc-binding peptide tethered to a rhodium intercalator
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DOI：
10.1021/ja9633981
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发表时间：
1997-04-09
影响因子：
15
通讯作者：
Barton, JK
中科院分区：
文献类型：
--
作者：
Fitzsimons, MP;Barton, JK
人工核酸酶的设计代表了一个非常感兴趣的领域。1虽然许多试剂已成功应用于RNA水解，2但由于DNA相对较高的水解稳定性，DNA的成功率较低3，4。5我们已经解决了通过结合DNA结合部分和反应部分来设计脱氧核糖核酸酶的问题：（i）以高亲和力结合在大沟中的铑嵌入剂6和（ii）模仿天然水解酶的束缚金属肽。7我们实验室之前的工作表明，与铑复合物相连的小肽以受附加肽控制的高位点特异性结合DNA。[8]在这里，我们扩展了这一策略，通过将锌促进的反应性并入拴系肽中来创建合成核酸内切酶。图1显示了我们的金属-肽缀合物Rh（phi）2bpy′-肽（Rh-P）的示意图。短肽Asp-Pro-Asp-Glu-Leu-Glu-His-Ala-Ala-Lys-His-Glu-Ala-Ala-Ala-Lys-CONH 2通过衍生自修饰的联吡啶配体的亚甲基接头与序列中性嵌入剂Rh（phi）2bpy′连接。含有菲醌二亚胺（phi）配体的铑（III）配合物结合在DNA的大沟中（Kd< 10-6 M）6，并且在光活化时在铑嵌入位点切割DNA。[10]基于含金属水解酶的活性位点，即在温和条件下发生生物聚合物水解的充分表征的金属酶位点，设计了与嵌入剂相连的肽。7将两个组氨酸沿着16个残基肽放置在位置7和11处，以在推定的肽的一面上产生锌配位位点
The design of artificial nucleases represents an area of substantial interest. 1 While many reagents have been successfully applied to RNA hydrolysis, 2 there have been fewer successes with DNA3, 4 due to its relatively high hydrolytic stability. 5 We have approached the problem of designing a deoxyribonuclease by combining both DNA-binding and reactive moieties:(i) a rhodium intercalator which binds in the major groove with high affinity6 and (ii) a tethered metallopeptide modeled after natural hydrolytic enzymes. 7 Previous work in our laboratory has shown that small peptides tethered to a rhodium complex bind DNA with high site specificity governed by the appended peptide. 8 Here we extend this strategy to create a synthetic endonuclease by incorporating zinc-promoted reactivity into the tethered peptide. 9 Figure 1 shows a schematic illustration of our metal-peptide conjugate, Rh (phi) 2bpy′-Peptide (Rh-P). A short peptide, Asp-Pro-Asp-Glu-Leu-Glu-His-Ala-Ala-Lys-His-Glu-Ala-Ala-Ala-Lys-CONH2, is tethered to Rh (phi) 2bpy′, a sequence-neutral intercalator, through a methylene linker derived from the modified bipyridine ligand. Complexes of rhodium (III) which contain a phenanthrenequinone diimine (phi) ligand bind in the major groove of DNA (Kd< 10-6 M) 6 and upon photoactivation cleave DNA at the rhodium intercalation site. 10 The peptide tethered to the intercalator was designed de noVo on the basis of the active sites of metal-containing hydrolases, wellcharacterized metalloenzyme sites in which biopolymer hydrolysis occurs under mild conditions. 7 Two histidines were placed in positions 7 and 11 along the 16-residue peptide to create a zinc coordination site on one face of a putative