Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA.
Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA.
复制标题
DOI：
10.1093/nar/gkn313
复制
发表时间：
2008-07
影响因子：
14.9
通讯作者：
Klug A
中科院分区：
文献类型：
--
作者：
Minczuk M;Papworth MA;Miller JC;Murphy MP;Klug A
突变线粒体DNA （mtDNA）分子的选择性降解是一种潜在的策略，可以用野生型（wt） mtDNA分子重新填充细胞，从而减轻mtDNA疾病的线粒体功能缺陷。锌指核酸酶（ZFNs）是一种与锌指肽（ZFP）结合的核酸酶，可结合特定的DNA序列，可用于选择性降解特定的mtDNA序列。通常，在目标DNA序列上使用异二聚化的互补zfn对；然而，传统的zfn在我们的系统中是无效的。为了克服这个问题，我们将两个FokI核酸酶结构域通过柔性连接物连接到具有n端线粒体靶向序列的ZFP上，从而创建了单链ZFP。在这里，我们发现这些ZFNs在细胞中被有效地转运到线粒体中，并以序列特异性的方式结合mtDNA，区分两个12bp的长序列，这些序列只相差一个碱基对。由于它们的选择性结合，它们在与突变相邻的预测位点上切割dsDNA。当在含有突变mtDNA和wt mtDNA混合的异质细胞中表达时，这些ZFNs选择性地降解突变mtDNA，从而增加细胞中wt mtDNA分子的比例。因此，线粒体靶向单链ZFNs是治疗mtDNA疾病的一种有希望的候选方法。
The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA molecules and thereby alleviate the defective mitochondrial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases conjugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the selective degradation of particular mtDNA sequences. Typically, pairs of complementary ZFNs are used that heterodimerize on the target DNA sequence; however, conventional ZFNs were ineffective in our system. To overcome this, we created single-chain ZFNs by conjugating two FokI nuclease domains, connected by a flexible linker, to a ZFP with an N-terminal mitochondrial targeting sequence. Here we show that these ZFNs are efficiently transported into mitochondria in cells and bind mtDNA in a sequence-specific manner discriminating between two 12-bp long sequences that differ by a single base pair. Due to their selective binding they cleave dsDNA at predicted sites adjacent to the mutation. When expressed in heteroplasmic cells containing a mixture of mutated and wt mtDNA these ZFNs selectively degrade mutated mtDNA, thereby increasing the proportion of wt mtDNA molecules in the cell. Therefore, mitochondria-targeted single-chain ZFNs are a promising candidate approach for the treatment of mtDNA diseases.
登录
查看更多内容
影响因子：
46.9
作者：
Isalan, M;Klug, A;Choo, Y
通讯作者：
Choo, Y
DOI：
10.1073/pnas.0609502103
发表时间：
2006-12-26
影响因子：
11.1
作者：
Minczuk, Michal;Papworth, Monika A.;Klug, Aaron
通讯作者：
Klug, Aaron
影响因子：
11
作者：
Tanaka, M;Borgeld, HJ;Yagi, K
通讯作者：
Yagi, K
影响因子：
3.3
作者：
Taylor, RW;Wardell, TM;Turnbull, DM
通讯作者：
Turnbull, DM
DOI：
10.1073/pnas.98.4.1437
发表时间：
2001-02-13
影响因子：
11.1
作者：
Moore, M;Klug, A;Choo, Y
通讯作者：
Choo, Y