Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein-staphylococcal nuclease hybrid.
Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein-staphylococcal nuclease hybrid.
复制标题
DOI：
10.1016/j.bbrc.2016.09.142
复制
发表时间：
2016-10
影响因子：
3.1
通讯作者：
Tomoaki Mori;Kento Nakamura;Keisuke Masaoka;Y. Fujita;Ryosuke Morisada;K. Mori;T. Tobimatsu;T. Sera
中科院分区：
文献类型：
--
作者：
Tomoaki Mori;Kento Nakamura;Keisuke Masaoka;Y. Fujita;Ryosuke Morisada;K. Mori;T. Tobimatsu;T. Sera
各种病毒感染动物和人类，并引起各种疾病，包括癌症。然而，预防病毒感染的有效方法尚未建立。因此，高度期望开发针对病毒的技术。我们已经证明，DNA病毒基因组的切割可以有效地阻止其复制。在这里，我们将这种方法扩展到RNA病毒。在本研究中，我们使用葡萄球菌核酸酶（SNase）代替人SMG 6的PIN结构域（PilT N-末端）作为RNA切割结构域，并将SNase融合到基于人Pumilio/fem-3结合因子（PUF）的人工RNA结合蛋白，以构建具有增强的流感病毒RNA切割速率的人工RNA限制性内切酶。所得的SNase融合核酸酶以比相应的PIN融合核酸酶高120倍的速率切割流感RNA。即使改变了PUF和RNA切割结构域之间的接头部分，PIN融合核酸酶的切割能力也没有提高。凝胶位移测定显示，所用的PUF衍生物的RNA结合性质不如野生型PUF。结合性质或设计方法的改进将允许SNase融合核酸酶切割哺乳动物细胞和/或生物体中的RNA靶标。
Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms.