A systematic study of several new types of hybrids of Cu-chelated clamped phenanthroline artificial metallonuclease (AMN) with triplex-forming oligonucleotides (TFO) for sequence-specific cleavage of double-stranded DNA (dsDNA) is reported. The synthesis of these AMN-TFO hybrids is based on application of the alkyne-azide cycloaddition click reaction as the key step. The AMN was attached through different linkers at either the 5'- or 3'-ends or in the middle of the TFO stretch. The diverse hybrids efficiently formed triplexes with the target purine-rich sequence and their copper complexes were studied for their ability to cleave dsDNA in the presence of ascorbate as a reductant. In all cases, the influence of the nature and length of the AMN-TFO, time, conditions and amounts of ascorbate were studied, and optimum conjugates and a procedure that gave reasonably efficient (up to 34 %) cleavage of the target sequence, while rendering an off-target dsDNA intact, were found. The footprint of cleavage on PAGE was identified only in one case, with low conversion; this means that cleavage does not proceed with single nucleotide precision. On the other hand, these AMN-TFO hybrids are useful for the selective degradation of target dsDNA sequences. Future improvements to this design may provide higher resolution and selectivity.

中文翻译：

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剪辑-邻菲咯啉三链体形成寡核苷酸杂交的氧化性DNA切割。

报道了对几种新型的铜螯合钳位菲咯啉人工金属核酸酶（AMN）与三链形成寡核苷酸（TFO）进行双链DNA（dsDNA）序列特异性切割的杂种的研究。这些AMN-TFO杂化物的合成是基于炔-叠氮化物环加成点击反应作为关键步骤的应用。AMN通过不同的连接子连接到5'或3'末端或TFO延伸的中间。各种杂种与富含嘌呤的靶序列有效地形成了三链体，并研究了它们的铜配合物在抗坏血酸作为还原剂存在下切割dsDNA的能力。在所有情况下，都研究了AMN-TFO的性质和长度，时间，条件和抗坏血酸含量的影响，找到了最佳结合物，并给出了可合理有效（高达34％）的靶序列裂解，同时使脱靶dsDNA完整的方法。仅在一种情况下发现了PAGE上裂解的足迹，转化率很低。这意味着切割不会以单核苷酸精度进行。另一方面，这些AMN-TFO杂种可用于目标dsDNA序列的选择性降解。对该设计的未来改进可能会提供更高的分辨率和选择性。这些AMN-TFO杂种可用于目标dsDNA序列的选择性降解。对该设计的未来改进可能会提供更高的分辨率和选择性。这些AMN-TFO杂种可用于目标dsDNA序列的选择性降解。对该设计的未来改进可能会提供更高的分辨率和选择性。