High-salt transcription from enzymatically gapped promoters nets higher yields and purity of transcribed RNAs.

T7 RNA polymerase is commonly used to synthesize large quantities of RNA for a wide variety of applications, from basic science to mRNA therapeutics. This in vitro system, while showing high fidelity in many ways, is also well known for producing longer than encoded RNA products, particularly under high-yield reaction conditions. Specifically, the resulting product pool is contaminated by an often disperse collection of longer cis-primed extension products. In addition to reducing yield via the conversion of correctly encoded RNA to longer products, self-primed extension generates partially double-stranded RNAs that can trigger the innate immune response. Extensive and low-yield purifications are then required to produce therapeutic RNA. Under high-yield conditions, accumulating concentrations of RNA effectively compete with promoter DNA for polymerase binding, driving self-primed extension at the expense of correct initiation. In the current work, we introduce a simple and novel modification in the DNA to strengthen promoter binding, shifting the balance back toward promoter-driven synthesis and so dramatically reducing self-primed extension. The result is higher yield of the encoded RNA at the outset and reduced need for extensive purifications. The approach can readily be applied to the synthesis of mRNA-length products under high-yield conditions. Introduction of a gap in the melted region of the promoter strengthens overall binding, allowing increased salt tolerance.

T7 RNA聚合酶通常用于合成大量的RNA，应用广泛，从基础科学到mRNA疗法均有涉及。这种体外系统虽然在很多方面表现出高保真度，但也因会产生比编码更长的RNA产物而闻名，特别是在高产反应条件下。具体而言，所得产物库常被一系列分散的较长顺式引物延伸产物所污染。除了因将正确编码的RNA转化为更长的产物而降低产量外，自身引物延伸还会产生部分双链RNA，从而引发先天免疫反应。因此需要进行大量且低产的纯化来生产治疗性RNA。在高产条件下，不断累积的RNA浓度会有效地与启动子DNA竞争聚合酶结合，从而以牺牲正确起始为代价驱动自身引物延伸。在当前的研究中，我们对DNA进行了一种简单而新颖的修饰，以增强启动子结合，使平衡重新向启动子驱动的合成倾斜，从而大幅减少自身引物延伸。其结果是一开始编码RNA的产量更高，并且对大量纯化的需求降低。该方法可轻易应用于高产条件下mRNA长度产物的合成。 在启动子的解链区域引入一个缺口可增强整体结合，提高耐盐性。