核糖核酸
生物
RNA沉默
抄写(语言学)
T7 RNA聚合酶
RNA依赖性RNA聚合酶
分子生物学
信使核糖核酸
DNA
聚合酶
细胞生物学
生物化学
RNA干扰
基因
大肠杆菌
噬菌体
语言学
哲学
作者
Kithmie MalagodaPathiranage,Ruptanu Banerjee,Craig T. Martin
摘要
Abstract Current approaches to RNA synthesis/manufacturing require substantial (and incomplete) purification post-synthesis. We have previously demonstrated the synthesis of RNA from a complex in which T7 RNA polymerase is tethered to promoter DNA. In the current work, we extend this approach to demonstrate an extremely stable system of functional co-tethered complex to a solid support. Using the system attached to magnetic beads, we carry out more than 20 rounds of synthesis using the initial polymerase-DNA construct. We further demonstrate the wide utility of this system in the synthesis of short RNA, a CRISPR guide RNA, and a protein-coding mRNA. In all cases, the generation of self-templated double stranded RNA (dsRNA) impurities are greatly reduced, by both the tethering itself and by the salt-tolerance that local co-tethering provides. Transfection of the mRNA into HEK293T cells shows a correlation between added salt in the transcription reaction (which inhibits RNA rebinding that generates RNA-templated extensions) and significantly increased expression and reduced innate immune stimulation by the mRNA reaction product. These results point in the direction of streamlined processes for synthesis/manufacturing of high-quality RNA of any length, and at greatly reduced costs.
科研通智能强力驱动
Strongly Powered by AbleSci AI