High-salt transcription of DNA cotethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA.

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.

通常采用两步法可获得高产率的RNA，即先用T7 RNA聚合酶进行高产率的体外转录，然后使用凝胶分离或色谱方法进行大量纯化。我们最近证明，在高产率的转录反应中，随着RNA在溶液中积累，T7 RNA聚合酶会重新结合并延伸编码的RNA（以RNA为模板），导致产物库受到比预期更长的（部分）双链杂质污染。目前的纯化方法往往无法完全去除这些杂质，如果它们存在于治疗药物中，可能会刺激先天免疫反应，从而带来潜在的致命后果。在这项工作中，我们引入了一种新的体外转录方法，该方法可产生高产率的编码RNA且无双链杂质，从而减少了进一步纯化的需求。转录在高盐条件下进行，以消除RNA产物的重新结合，同时将启动子DNA和T7 RNA聚合酶在磁珠上紧密共连接，以驱动启动子结合和转录起始，从而仅使编码RNA的总产量和纯度提高。在合成过程中更彻底地去除双链RNA不仅会降低总体生产成本，而且最终应该能够使目前受这些杂质阻碍的治疗方法和技术得以实现。