Abdelhady Ahmed Mostafa, Hirano Yu, Onizuka Kazumitsu, Okamura Hidenori, Komatsu Yasuo, Nagatsugi Fumi
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi, Japan.
Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Miyagi, Japan.
Curr Protoc. 2022 Mar;2(3):e386. doi: 10.1002/cpz1.386.
Crosslinking reactions to nucleic acids are an effective way to prepare stable complexes formed by covalent bonding. We demonstrated that fully 2'-O-methylated (2'-OMe) RNAs having a 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. Recently, we reported the preparation of crosslinked 2'-OMe RNA duplexes using AVP and the anti-miRNA oligonucleotides (AMOs) containing crosslinked duplexes at the terminal positions. These AMOs exhibited efficient microRNA (miRNA) inhibition at very low concentrations. In this article, we describe the chemical synthesis of 2'-OMe oligonucleotides containing AVP and preparation of the AMOs bearing crosslinked 2'-OMe RNA duplexes using AVP. In addition, we describe in detail the miRNA inhibition assay using these AMOs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphoramidite of 2-amino-6-vinylguanosine derivative Basic Protocol 2: Synthesis of AVP-2'-OMe RNA Basic Protocol 3: Evaluation of the crosslink reactivity of CFO containing AVP to the 2'-OMe RNA and preparation of AMOs containing crosslinked duplex Basic Protocol 4: miRNA inhibition assays.
与核酸的交联反应是制备通过共价键形成的稳定复合物的有效方法。我们证明，具有2-氨基-6-乙烯基嘌呤（AVP）的完全2'-O-甲基化（2'-OMe）RNA对靶RNA中的尿嘧啶表现出有效的交联作用。最近，我们报道了使用AVP制备交联的2'-OMe RNA双链体以及在末端位置含有交联双链体的抗miRNA寡核苷酸（AMO）。这些AMO在非常低的浓度下表现出高效的微小RNA（miRNA）抑制作用。在本文中，我们描述了含AVP的2'-OMe寡核苷酸的化学合成以及使用AVP制备带有交联2'-OMe RNA双链体的AMO。此外，我们详细描述了使用这些AMO的miRNA抑制试验。© 2022威利期刊有限责任公司。基本方案1：2-氨基-6-乙烯基鸟苷衍生物亚磷酰胺的合成 基本方案2：AVP-2'-OMe RNA的合成 基本方案3：含AVP的CFO与2'-OMe RNA的交联反应性评估及含交联双链体的AMO的制备 基本方案4：miRNA抑制试验。
