College of Food Science and Engineering, Ocean University of China, Qingdao 266550, Shandong, China.
Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266235, Shandong, China.
RNA. 2023 Nov;29(11):1691-1702. doi: 10.1261/rna.079670.123. Epub 2023 Aug 3.
Double-stranded RNA (dsRNA) has aroused widespread interest due to its effects on immunity and applications based on RNAi. However, the in vitro preparation of dsRNA is costly and laborious. In this study, we have developed a novel and interesting method designated as pfRCT (promoter-free rolling-circle transcription) for direct, facile, and efficient dsRNA preparation. This method generates equal amounts of sense and antisense strands simultaneously from a single circular dsDNA template. To initiate transcription by T7 RNA polymerase without directional preference, a 9-15-bp bubble (mismatched duplex with strong sequence symmetry) is introduced into the template. During RCT, all the necessary reagents, including the template, NTPs, RNA polymerase, RNase H, and Helpers, are present in one pot; and the just-transcribed RNA is immediately truncated by RNase H to monomers with the desired size. The ends of the dsRNA product can also be simply sealed by T4 RNA ligase 1 after pfRCT. This new approach is expected to promote the applications of dsRNA.
双链 RNA（dsRNA）因其对免疫的影响和基于 RNAi 的应用而引起了广泛的关注。然而，dsRNA 的体外制备既昂贵又费力。在这项研究中，我们开发了一种新颖有趣的方法，称为 pfRCT（无启动子滚环转录），用于直接、简便、高效地制备 dsRNA。该方法从单个环形 dsDNA 模板同时生成等量的正义和反义链。为了在没有方向性偏好的情况下由 T7 RNA 聚合酶起始转录，在模板中引入了 9-15 个碱基的泡（具有强序列对称性的错配双链）。在 RCT 过程中，所有必要的试剂，包括模板、NTPs、RNA 聚合酶、RNase H 和 Helper，都在一个管中；并且刚刚转录的 RNA 立即被 RNase H 截断成所需大小的单体。dsRNA 产物的末端在 pfRCT 后也可以通过 T4 RNA 连接酶 1 简单封闭。这种新方法有望促进 dsRNA 的应用。
