在Riboswitch启发的Aptamers的体外选择和体内测试
Michael G Mohsen1,2, Ronald R Breaker1,2,3
1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA.
Bio-protocol
|July 17, 2023
概括
通过体外选择开发用于新配体的新型 рибо开关启发的吸收体. 这种方法通过利用天然的RNA支架,在复杂的细胞环境中增强了体功能.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 合成生物学 合成生物学
背景情况:
- 在实验室中对工程性体的选择方法可能会产生在细胞环境中有效性有限的分子.
- 天然存在的核糖开关阿马提供结构性支架,可以改善体外衍生阿马的性能.
- 在生物技术应用中,弥合体外受体选择和细胞功能之间的差距至关重要.
研究的目的:
- 提供一个全面的指南,用于设计和实验开发 riboswitch-inspired aptamers 的新型连接体.
- 适应和优化Capture-SELEX协议,以生成具有增强细胞兼容性的aptamers.
- 建立可靠的方法来验证从初始选择到细菌细胞中的功能测试的aptamer候选物.
主要方法:
- 使用Capture-SELEX与含有riboswitch aptamer支架的RNA池进行体外选择.
- 采用策略来缓解在选择过程中自私序列的主导地位.
- 利用下一代测序和生物信息学来识别aptamer候选物.
- 在细菌细胞培养物中进行aptamer候选物的生物化学验证和功能测试.
主要成果:
- 证明了一个可通用的体外选择协议,可适应各种RNA支架和多重连接体候选物.
- 成功识别和验证了具有细胞应用潜力的新型吸收酶候选物.
- 通过重新移植到表达平台,在细菌细胞上下文中建立了一个测试aptamer功能框架.
结论:
- 使用体外选择的Riboswitch启发的aptamer开发是一种可行的策略,用于创建复杂生物系统的功能分子.
- 提出的Capture-SELEX协议和验证管道对于发现和测试新体是有效的.
- 这种方法为各种生物技术和治疗应用的基于RNA的工程工具提供了一个有希望的途径.
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