Site-specific RNA functionalization is in high demand, but remains a challenge, particularly for RNAs produced by transcription rather than by total synthesis. Recent studies have described acylimidazole reagents that react in high yields at 2'-OH groups in RNAs. To date, the reactions occur stochastically at non-base-paired regions of RNA, covering much of the RNA in scattered acyl esters. Localized reactions, if possible, could prove useful in many applications, providing functional handles at specific sites and sequences of the biopolymer. Here we describe a DNA- directed strategy for in vitro functionalization of RNA at site-localized 2'-OH groups. The method, RNA Acylation at Induced Loops (RAIL), utilizes complementary helper DNA oligonucleotides that expose gaps or loops at selected positions while protecting the remainder in DNA-RNA duplexes. Reaction with acylimidazole reagents is then carried out, providing high yields of 2'-OH conjugation at predetermined sites. Subsequent removal of the DNA provides the RNA functionalized as desired. Experiments reveal optimal helper oligodeoxynucleotide designs and conditions for the reaction, and tests of the approach were carried out to control ribozyme activities and to label RNAs with dual-color fluorescent dyes. The RAIL approach offers a simple new strategy for site-specific labeling and controlling RNAs of any length and origin.

中文翻译：

---

通过DNA诱导的结构进行位点特异性RNA功能化

对位点特异性RNA的功能化有很高的要求，但仍然是一个挑战，特别是对于通过转录而非全合成产生的RNA。最近的研究描述了酰基咪唑试剂，它们在RNA的2'-OH基团上以高收率反应。迄今为止，反应随机发生在RNA的非碱基配对区域，覆盖了分散的酰基酯中的许多RNA。如果可能的话，局部反应可能会在许多应用中证明是有用的，可以在生物聚合物的特定位点和序列上提供功能性处理。在这里，我们描述了针对DNA的体外定向2'-OH基团体外功能化的策略。该方法，“诱导环RNA酰化”（RAIL），利用互补的辅助DNA寡核苷酸，可暴露选定位置的缺口或环，同时保护DNA-RNA双链体中的其余部分。然后与酰基咪唑试剂进行反应，从而在预定位点提供高产率的2'-OH共轭。随后去除DNA可提供所需功能的RNA。实验揭示了最佳的辅助寡聚脱氧核苷酸设计和反应条件，并对该方法进行了测试，以控制核酶活性并用双色荧光染料标记RNA。RAIL方法为针对特定长度的标记和控制任何长度和来源的RNA提供了一种简单的新策略。随后去除DNA可提供所需功能的RNA。实验揭示了最佳的辅助寡聚脱氧核苷酸设计和反应条件，并对该方法进行了测试，以控制核酶活性并用双色荧光染料标记RNA。RAIL方法提供了一种简单的新策略，用于特定位置的标记和控制任何长度和来源的RNA。随后去除DNA可提供所需功能的RNA。实验揭示了最佳的辅助寡聚脱氧核苷酸设计和反应条件，并对该方法进行了测试，以控制核酶活性并用双色荧光染料标记RNA。RAIL方法为针对特定长度的标记和控制任何长度和来源的RNA提供了一种简单的新策略。