RNA 5′叠氮化物的固相合成及其在标记、连接和环化中的应用

Q4 Chemistry
Marcin Warminski, Joanna Kowalska, Jacek Jemielity
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引用次数: 4

摘要

具有5 '官能团的rna作为分子探针和报告分子已引起人们的兴趣。铜催化叠氮化物-炔环加成是获得此类分子最直接的方法之一;然而,叠氮基团的RNA功能化一直是合成上的挑战。本文介绍了一种简单有效的固相低核苷酸5′端叠氮化物功能化方法。叠氮化物部分通过两个步骤直接连接到C5 '端:(i)使用甲基三苯氧磷碘化将-OH转化为-I,以及(ii)使用叠氮化物钠将-I转化为-N3。通过荧光标记铜(I)催化(CuAAC)和菌株促进(SPAAC)叠氮化物-炔环加成反应,两个RNA片段的连接以及短双功能化寡核苷酸的环化,可以证明所得化合物的反应性。该方案利用固体载体上的标准磷酸酰胺方法合成的寡核苷酸,使用市售的2 ' - o - pivm保护单体。这种保护策略消除了磷酰胺法合成RNA中常用的碘化试剂与硅基保护基团(TBDMS、TOM)之间的干扰。©2020 Wiley期刊有限公司。基本方案1:固相合成低核糖核苷酸5 ' -叠氮化物基本方案2:在溶液中标记低核糖核苷酸5 ' -叠氮化物备选方案1:在固体载体上标记低核糖核苷酸5 ' -叠氮化物基本方案3:低核糖核苷酸5 ' -叠氮化物的空间标记基本方案4:低核糖核苷酸5 ' -叠氮化物的空间标记基本方案5:低核糖核苷酸5 ' -叠氮化物的CuAAC环化基本方案5:低核糖核苷酸5 ' -叠氮化物的CuAAC环化支持方案:HPLC纯化
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Solid-Phase Synthesis of RNA 5′-Azides and Their Application for Labeling, Ligation, and Cyclization Via Click Chemistry

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 -N3 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

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来源期刊
Current Protocols in Nucleic Acid Chemistry
Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry
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期刊介绍: Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.
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