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[1]周姗 蒲沁琳 黄鑫 袁奕 杜凤 董娟 陈刚毅 崔欣 唐卓**.通过理性设计在体内获得具有分子间剪切潜力的锤头型适体酶[J].应用与环境生物学报,2021,27(02):1-13.[doi:10.19675/j.cnki.1006-687x.2020.03049]
 ZHOU Shan,PU Qinlin,et al.Obtaining hammerhead aptazymes with trans-cleaving potential in vivo by rational design[J].Chinese Journal of Applied & Environmental Biology,2021,27(02):1-13.[doi:10.19675/j.cnki.1006-687x.2020.03049]
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通过理性设计在体内获得具有分子间剪切潜力的锤头型适体酶()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
27卷
期数:
2021年02期
页码:
1-13
栏目:
研究论文
出版日期:
2021-04-25

文章信息/Info

Title:
Obtaining hammerhead aptazymes with trans-cleaving potential in vivo by rational design
作者:
周姗12 蒲沁琳12 黄鑫1 袁奕1 杜凤1 董娟1 陈刚毅1 崔欣1 唐卓1**
1中国科学院成都生物研究所 成都 610041
2中国科学院大学 北京 100049
Author(s):
ZHOU Shan1 2 PU Qinlin1 2 Huang Xin1 Yuan Yi1 Du Feng1 Dong Juan1 Chen Gangyi1 Cui Xin1 & TANG Zhuo1**
1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2 University of Chinese Academy of Sciences, Beijing 100049, China
关键词:
锤头型核酶锤头型适体酶茶碱分子间剪切基因治疗
Keywords:
hammerhead ribozyme hammerhead aptazyme theophylline trans-cleaving gene therapy
DOI:
10.19675/j.cnki.1006-687x.2020.03049
摘要:
锤头型适体酶目前已广泛用于基因表达调控,为了维持锤头型核酶高效自剪切的Loop I 和Loop II 之间的三级结构互作,现有的锤头型适体酶都是基于其stem III或stem I 发展而来。为增加锤头型适体酶的多样性和通用性,探索基于stem II的锤头型适体酶,通过理性设计基于stem II的锤头型适体酶的链接体,基于毒蛋白ibsC报告体系,结合宿主的生长推测锤头型适体酶响应小分子调控的情况。设计了10种不同长度、不同碱基互补配对程度的链接体。其中7种链接体对应的锤头型适体酶无法发生自剪切,2种可以发生自剪切,但是不受茶碱调控。最终获得一个基于stem II的锤头型适体酶T4,其链接体由4对完全互补配对碱基对组成,这表明由4对完全互补配对碱基对组成的链接体可能更容易获得小分子依赖的锤头型适体酶。在茶碱存在时,含有T4的宿主生长,而茶碱不存在时,其宿主不能生长,这表明锤头型适体酶T4的自剪切活性是受茶碱调控的,成功实现了利用小分子控制大肠杆菌中毒蛋白基因的表达。综上,T4是基于stem II 的依赖茶碱的锤头型适体酶,表明以锤头型核酶的stem II 也可以发展锤头型适体酶;此外,T4具有分子间剪切潜力,有望发展成为利用小分子调控内源性基因表达的工具。 (图7 表2 参39)
Abstract:
Hammerhead aptazymes have been widely used to regulate gene expression, in order to maintain the tertiary structure interaction between Loop I and Loop II which is vital to highly efficient self-cleavage, the existing hammerhead aptazymes are based on stem III or stem I, and hammerhead aptazymes based on stem II have not been reported. To increase the d iversity and universality of hammerhead aptazymes, we explored hammerhead aptazymes based on stem II. Through rationally designed the communication module of hammerhead aptazymes based on stem II , and based on the toxic protein ibsC reporting system, and combined with the growth of the host , we speculate how hammerhead aptazymes respond to the regulation of small molecules. In t his study, 10 types of communication modules with different lengths and different base complementary pairing degrees were designed. Among them, the hammerhead aptazymes corresponding to 7 kinds of communication modules couldn’t self-cleave, and 2 kinds could self-cleave, but they were not regulated by theophylline. Finally we obtained a hammerhead aptazyme T4 based on stem II, whose communication module consists of four pairs of completely complementary base pairs, which indicated that communication module consists of four pairs of completely complementary base pairs might be easier to obtain small molecules-dependent hammerhead aptazymes. In the presence of theophylline, the host including T4 grew, while in the absence of theophylline, the host could not grow, which indicated that the self-cleaving activity of hammerhead aptazyme T4 was regulated by theophylline, and successfully achieved that using small molecules to control gene expression in Escherichia coli. T4 is a theophylline-dependent hammerhead aptazyme based on stem II, which indicates that we could develop hammerhead aptazymes based on stem II. Besides, with the trans-cleaving potential , T4 is expected to develop into a novel tool that uses small molecules to regulate the expression of endogenous genes.

备注/Memo

备注/Memo:
收稿日期 Received: 2020-03-25 接受日期 Accepted: 2020-05-06
中国国家自然科学基金会(21322208;21572222)、中国科学院西部之光基金(Y4C3021100)、四川省科学技术项目基金((2014RZ0022) Supported by National Natural Sciences Foundation of China (Grant No. 21322208 and 21572222); West Light Foundation of The Chinese Academy of Sciences (Y4C3021100); Science and technology project of Sichuan Province (2014RZ0022).
**通讯作者 Corresponding author (E-mail: tangzhuo@cib.ac.cn)
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更新日期/Last Update: 2020-05-22