使用HUH标签在单个步骤中进行序列定向的共价蛋白-DNA链接
Klaus N Lovendahl1, Amanda N Hayward1, Wendy R Gordon1
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Journal of the American Chemical Society
|May 9, 2017
概括
我们开发了HUH标签, 这些标签与DNA序列进行特定反应,使基因编辑,单分子研究和细胞成像中的应用成为可能.
科学领域:
- 生物化学
- 分子生物学
- 化学生物学
背景情况:
- 共价蛋白-DNA链接对于各种生物过程和生物技术应用至关重要.
- 现有的蛋白质-DNA结合方法通常需要修改的DNA或缺乏特异性.
- 在生物系统中需要新的蛋白质-DNA结合策略.
研究的目的:
- 开发一种强大的多功能方法来对蛋白质和DNA进行共价连接.
- 为特定的DNA序列识别识别和表征HUH内核酶域作为融合伙伴 (HUH标签).
- 在基因编辑,单分子研究和细胞成像中展示HUH标签的实用性.
主要方法:
- 设计HUH内核酶域作为融合蛋白 (HUH标签).
- 用未经修改的单链DNA序列测试HUH标签的反应性.
- 识别可以识别不同的DNA序列的直角HUH标签.
- 将HUH标签集成到Cas9介导的基因编辑系统中.
- 构建双重结合的蛋白质用于单分子实验.
- 在活细胞和固定细胞中应用HUH标签.
主要成果:
- HUH标签与特定的,未经修改的单链DNA序列具有强大的共价链接.
- 鉴定出五种不同的HUH标签,它们具有不同DNA序列的正交反应性.
- 在Cas9基因编辑中成功使用HUH标签作为融合伙伴.
- 为了先进的单分子研究, 构建了双重结合的蛋白质.
- 该方法在活细胞和固定细胞中的细胞成像应用中得到验证.
结论:
- HUH标签为共价蛋白-DNA结合提供了多功能和强大的平台.
- 已识别的HUH标签的直角性扩大了它们在复杂的生物系统中的有用性.
- HUH标签技术为基因编辑,单分子生物物理学和活细胞成像提供了显著的潜力.
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