Solid‐Phase Synthesis of RNA 5′‐Azides and Their Application for Labeling, Ligation, and Cyclization Via Click Chemistry

Abstract

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.