合成生物学
生物传感器
计算生物学
核糖核酸
RNA聚合酶
纳米技术
聚合酶
T7 RNA聚合酶
定向进化
生物物理学
生物
细胞生物学
DNA
基因
遗传学
生物化学
噬菌体
材料科学
大肠杆菌
突变体
作者
Jinyue Pu,Julia Zinkus-Boltz,Bryan C. Dickinson
标识
DOI:10.1038/nchembio.2299
摘要
Design of a proximity-dependent split RNA polymerase system and its optimization by phage-assisted continuous evolution (PACE) enabled the development of a family of activity-dependent split RNA polymerase biosensors regulated by small molecules or light. Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein–protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small molecules, demonstrating the 'plug-and-play' nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein–protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.
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