Toward a general approach for RNA-templated hierarchical assembly of split-proteins.
Toward a general approach for RNA-templated hierarchical assembly of split-proteins.
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DOI：
10.1021/ja104395b
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发表时间：
2010-08-25
影响因子：
15
通讯作者：
Ghosh, Indraneel
中科院分区：
文献类型：
--
作者：
Furman, Jennifer L.;Badran, Ahmed H.;Ajulo, Oluyomi;Porter, Jason R.;Stains, Cliff I.;Segal, David J.;Ghosh, Indraneel
在存在用户定义的 RNA 靶标的情况下有条件地打开信号或诱导功能的能力在医学和合成生物学中具有潜在的应用。尽管序列特异性 pumilio 重复蛋白可以靶向一组有限的 ssRNA 序列，但没有通用方法可以用设计的蛋白靶向 ssRNA。作为 RNA 识别的第一步，我们利用参与 RNA 干扰的 argonaute RNA 结合域，专门针对任何 dsRNA 的通用 2 核苷酸、3' 突出端。当 ssRNA 被设计的互补引导序列靶向时，我们测试了由 argonute 介导的对新生成的核苷酸突出端的识别引导的分裂荧光素酶的重新组装。当 argonaute 与 pumilio 重复序列结合使用时，这种方法取得了成功，并扩大了潜在 ssRNA 靶标的范围。然而，靶向任何所需的 ssRNA 仍然难以捉摸，因为两个 argonute 结构域提供了最少的重组分裂荧光素酶。接下来，我们设计并测试了第二个分层组装，其中 ssDNA 引导附加到 DNA 发夹，作为附着到分裂荧光素酶的高亲和力锌指的支架。当存在含有与导向互补的相邻序列的 ssRNA 靶标时，发夹会靠近，从而允许锌指结合并伴随片段化荧光素酶的重新组装。这种新方法的范围通过专门针对编码 VEGF、hDM2 和 HER2 的 RNA 进行了验证。这些方法为在任何所需 ssRNA 靶标存在的情况下片段化蛋白质的条件重组提供了潜在的通用设计范例。
The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3' overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target.
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影响因子：
11.4
作者：
Elbashir, SM;Martinez, J;Tuschl, T
通讯作者：
Tuschl, T
DOI：
10.1073/pnas.0610877104
发表时间：
2007-02-13
影响因子：
11.1
作者：
Chelur, Dattananda S.;Chalfie, Martin
通讯作者：
Chalfie, Martin
影响因子：
12.4
作者：
Alwin, S;Gere, MB;Cathomen, T
通讯作者：
Cathomen, T
影响因子：
46.9
作者：
Galarneau, A;Primeau, M;Michnick, SW
通讯作者：
Michnick, SW
影响因子：
15
作者：
Deng, Minggang;Zhang, Dan;Zhou, Xiang
通讯作者：
Zhou, Xiang