Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units
Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units
复制标题
DOI：
10.1073/pnas.98.4.1437
复制
发表时间：
2001-02-13
影响因子：
11.1
通讯作者：
Choo, Y
中科院分区：
文献类型：
--
作者：
Moore, M;Klug, A;Choo, Y
在寻找体内应用如基因治疗的“理想”设计转录因子方面，多肽指状肽可能会变得更加突出。然而，为了使这些治疗有效和安全，这些多肽必须与高亲和力结合，更重要的是，必须具有极高的特异性。我们之前的研究表明，锌指阵列可以与18bp的DNA结合，具有皮摩尔亲和力，但也表明手指阵列也可能与相关序列紧密结合。这项工作解决了锌指DNA结合的特异性问题。我们表明，通过改变锌指阵列的构建方式--通过连接三个双指结构域而不是两个三指单元--可以通过增加对突变或密切相关序列的辨别来实现更高的靶标特异性。这些新的多肽具有额外的能力，能够跨越两个未结合的DNA的短间隙，尽管仍然与它们的目标位置具有皮摩尔亲和力。我们相信，这种构建锌指阵列的新方法在基因治疗和转基因生物生产方面将比以前报道的锌指阵列提供更大的效果。
Multizinc finger peptides are likely to reach increased prominence in the search for the "ideal" designer transcription factor for in vivo applications such as gene therapy. However, for these treatments to be effective and safe, the peptides must bind with high affinity and, more importantly, with great specificity. Our previous research has shown that zinc finger arrays can be made to bind 18 bp of DNA with picomolar affinity, but also has suggested that arrays of fingers also may bind tightly to related sequences. This work addresses the question of zinc finger DNA binding specificity. We show that by changing the way in which zinc finger arrays are constructed-by linking three two-finger domains rather than two three-finger units-far greater target specificity can be achieved through increased discrimination against mutated or closely related sequences. These new peptides have the added capability of being able to span two short gaps of unbound DNA, although still binding with picomolar affinity to their target sites. We believe that this new method of constructing zinc finger arrays will offer greater efficacy in the fields of gene therapy and in the production of transgenic organisms than previously reported zinc finger arrays.