Department of Chemistry, ChEM-H Institute and Stanford Cancer Institute, Stanford University, Stanford, California 94305, United States.
J Am Chem Soc. 2020 Sep 23;142(38):16357-16363. doi: 10.1021/jacs.0c06824. Epub 2020 Sep 14.
Methods for RNA functionalization at specific sites are in high demand but remain 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 stochastically at nonbase-paired regions, 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 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 an acylimidazole reagent is then carried out, providing high yields of 2'-OH conjugation at predetermined sites. Experiments reveal optimal helper oligodeoxynucleotide designs and conditions for the reaction, and tests of the approach are carried out to control localized ribozyme activities and to label RNAs with dual-color fluorescent dyes. The RAIL approach offers a simple and novel strategy for site-selective labeling and control of RNAs, potentially of any length and origin.
方法的 RNA 功能化在特定的网站是在高需求，但仍然是一个挑战，尤其是对于 RNA 转录产生的而不是通过总合成。最近的研究已经描述酰基咪唑试剂反应在高产率在 2 ' -羟基组随机在非碱基配对区，覆盖了大部分的 RNA 在分散的酰基酯。局部反应，如果可能的话，可能会在许多应用中证明是有用的，提供功能处理的特定网站和序列的生物聚合物。在这里，我们描述了一个 DNA 指导的策略，为功能化的 RNA 在局部 2 ' -羟基组。该方法，RNA 酰化诱导环（铁路），利用互补的辅助 DNA 寡核苷酸，暴露的差距或循环在选定的位置，同时保护其余的在 DNA - RNA 双链。反应与酰基咪唑试剂，然后进行，提供高产量的 2 ' -羟基共轭在预定的网站。实验揭示了最佳的辅助寡脱氧核苷酸设计和反应条件，并进行了测试的方法来控制局部核酶活性和标记的 RNA 与双荧光染料。铁路的方法提供了一个简单和新颖的策略，用于选择性标记和控制 RNA ，可能是任何长度和来源。
