Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase
Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase
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DOI：
10.1073/pnas.0609502103
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发表时间：
2006-12-26
影响因子：
11.1
通讯作者：
Klug, Aaron
中科院分区：
文献类型：
--
作者：
Minczuk, Michal;Papworth, Monika A.;Klug, Aaron
我们使用工程锌指微粒（ZFPs）选择性地结合人类mtDNA中的预定序列。令人惊讶的是，我们发现仅使用传统的线粒体靶向序列，工程ZFPs不能可靠地传递到线粒体。我们在这里表明，添加核输出信号允许锌指嵌合酶输入到人类线粒体。线粒体特异性ZFPs与mtDNA的选择性结合以靶向T8993G突变为例，该突变可导致两种线粒体疾病，神经源性肌无力、共济失调和视网膜色素变性（NARP），以及母体遗传性Leigh综合征。为了开发一种能够监测mtDNA位点特异性改变的系统，我们将UP与易于测定的hDNMT3a甲基化酶的dna修饰活性结合起来。突变特异性嵌合甲基化酶的表达导致突变位点附近的胞嘧啶选择性甲基化。这证明了利用锌指技术以序列特异性的方式靶向和改变mtDNA是可能的。
We used engineered zinc finger pepticles (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a UP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology.