抄写(语言学)
聚合酶
RNA依赖性RNA聚合酶
核糖核酸
RNA聚合酶
化学
RNA聚合酶Ⅰ
DNA
T7 RNA聚合酶
RNA聚合酶Ⅱ
分子生物学
生物
生物化学
基因
基因表达
发起人
噬菌体
大肠杆菌
哲学
语言学
作者
Elvan Cavac,Luis E. Ramírez-Tapia,Craig T. Martin
标识
DOI:10.1016/j.jbc.2021.100999
摘要
High yields of RNA are routinely prepared following the two-step approach of high-yield in vitro transcription using T7 RNA polymerase followed by extensive purification using gel separation or chromatographic methods. We recently demonstrated that in high-yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer-than-desired, (partially) double-stranded impurities. Current purification methods often fail to fully eliminate these impurities, which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. In this work, we introduce a novel in vitro transcription method that generates high yields of encoded RNA without double-stranded impurities, reducing the need for further purification. Transcription is carried out at high-salt conditions to eliminate RNA product rebinding, while promoter DNA and T7 RNA polymerase are cotethered in close proximity on magnetic beads to drive promoter binding and transcription initiation, resulting in an increase in overall yield and purity of only the encoded RNA. A more complete elimination of double-stranded RNA during synthesis will not only reduce overall production costs, but also should ultimately enable therapies and technologies that are currently being hampered by those impurities.
科研通智能强力驱动
Strongly Powered by AbleSci AI