Warminski Marcin, Kowalska Joanna, Jemielity Jacek
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland.
Centre of New Technologies, University of Warsaw, Warsaw, Poland.
Curr Protoc Nucleic Acid Chem. 2020 Sep;82(1):e112. doi: 10.1002/cpnc.112.
RNAs with 5' functional groups have been gaining interest as molecular probes and reporter molecules. Copper-catalyzed azide-alkyne cycloaddition is one of the most straightforward methods to access such molecules; however, RNA functionalization with azide group has been posing a synthetic challenge. This article describes a simple and efficient protocol for azide functionalization of oligoribonucleotides 5'-end in solid-phase. An azide moiety is attached directly to the C5'-end in two steps: (i) -OH to -I conversion using methyltriphenoxyphosphonium iodide, and (ii) -I to -N substitution using sodium azide. The reactivity of the resulting compounds is exemplified by fluorescent labeling using both copper(I)-catalyzed (CuAAC) and strain-promoted (SPAAC) azide-alkyne cycloaddition reactions, ligation of two RNA fragments, and cyclization of short bifunctionalized oligonucleotides. The protocol makes use of oligoribonucleotides synthesized by standard phosphoramidite approach on solid support, using commercially available 2'-O-PivOM-protected monomers. Such a protection strategy eliminates the interference between the iodination reagent and silyl protecting groups (TBDMS, TOM) commonly used in RNA synthesis by phosphoramidite approach. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Solid-phase synthesis of oligoribonucleotide 5'-azides Basic Protocol 2: CuAAC labeling of oligoribonucleotide 5'-azides in solution Alternate Protocol 1: CuAAC labeling of oligoribonucleotide 5'-azides on solid support Basic Protocol 3: SPAAC labeling of oligoribonucleotide 5'-azides Basic Protocol 4: CuAAC ligation of oligoribonucleotide 5'-azides Basic Protocol 5: CuAAC cyclization of oligoribonucleotide 5'-azides Support Protocol: HPLC Purification.
具有5'官能团的RNA作为分子探针和报告分子越来越受到关注。铜催化的叠氮化物-炔烃环加成反应是获得此类分子最直接的方法之一；然而，用叠氮基团对RNA进行功能化一直是一个合成挑战。本文描述了一种在固相中将寡核糖核苷酸5'-末端进行叠氮功能化的简单有效方案。通过两步将叠氮部分直接连接到C5'-末端：（i）使用甲基三苯氧基碘化鏻将-OH转化为-I，以及（ii）使用叠氮化钠进行-I到-N的取代。所得化合物的反应活性通过使用铜（I）催化的（CuAAC）和应变促进的（SPAAC）叠氮化物-炔烃环加成反应进行荧光标记、两个RNA片段的连接以及短双功能化寡核苷酸的环化来举例说明。该方案利用通过标准亚磷酰胺方法在固相上合成的寡核糖核苷酸，使用市售的2'-O-PivOM保护的单体。这种保护策略消除了碘化试剂与亚磷酰胺方法合成RNA中常用的硅烷基保护基团（TBDMS、TOM）之间的干扰。©2020威利期刊有限责任公司。基本方案1：寡核糖核苷酸5'-叠氮化物的固相合成；基本方案2：溶液中寡核糖核苷酸5'-叠氮化物的CuAAC标记；替代方案1：固相上寡核糖核苷酸5'-叠氮化物的CuAAC标记；基本方案3：寡核糖核苷酸5'-叠氮化物的SPAAC标记；基本方案4：寡核糖核苷酸5'-叠氮化物的CuAAC连接；基本方案5：寡核糖核苷酸5'-叠氮化物的CuAAC环化；支持方案：HPLC纯化。
