Rings of single-stranded RNA are promising for many practical applications, but the methods to prepare them in preparative scale have never been established. Previously, RNA circularization was achieved by T4 RNA ligase 2 (Rnl2, a dsRNA ligase) using splints, but the yield was low due to concurrent intermolecular polymerization. Here, various functional RNAs (siRNA, miRNA, ribozyme, etc.) are dominantly converted by Rnl2 to the rings without significant limitations in sizes and sequences. The key is to design a precursor RNA, which is highly activated for the efficient circularization without any splint. First, secondary structure of target RNA ring is simulated by Mfold, and then hypothetically cut at one site so that a few intramolecular base pairs are formed at the terminal. Simply by treating this RNA with Rnl2, the target ring was selectively and efficiently produced. Unexpectedly, circular RNA can be obtained in high yield (>90%), even when only 2 bp form in the 3'-OH side and no full match base pair forms in the 5'-phosphate side. Formation of polymeric by-products was further suppressed by diluting conventional Rnl2 buffer to abnormally low concentrations. Even at high-RNA concentrations (e.g. 50 μM), enormously high selectivity (>95%) was accomplished.

中文翻译：

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通过前体链的合理设计，无需任何夹板，T4 RNA连接酶2优先产生RNA环。


单链RNA环在许多实际应用中都有希望，但制备规模制备它们的方法尚未建立。此前，RNA环化是通过T4 RNA连接酶2（Rnl2，一种dsRNA连接酶）使用夹板实现的，但由于同时发生分子间聚合，产量较低。在这里，各种功能性RNA（siRNA、miRNA、核酶等）主要由Rnl2转化为环，在大小和序列上没有显着限制。关键是设计一种高度活化的前体RNA，无需任何夹板即可实现高效环化。首先，通过Mfold模拟靶RNA环的二级结构，然后在一个位点进行假设切割，以便在末端形成一些分子内碱基对。只需用 Rnl2 处理该 RNA，即可选择性且高效地产生目标环。出乎意料的是，即使 3'-OH 侧仅形成 2 bp 并且 5'-磷酸侧没有形成完全匹配的碱基对，也能以高产率 (>90%) 获得环状 RNA。通过将常规 Rnl2 缓冲液稀释至异常低浓度，进一步抑制了聚合物副产物的形成。即使在高 RNA 浓度（例如 50 μM）下，也能实现极高的选择性（>95%）。