Abstract

Although the modern biological systems are by large based on DNA genomes and protein enzymes, RNA plays important roles in many fundamental processes in cells, including regulation of protein biosynthesis, RNA splicing, and retroviral replication, with remarkable features. From this point of view, site-specific substitution and derivatization of RNA can provide powerful tools for elucidating RNA structures and functions. The modification of either the 3'and 5'-termini or an internal position of the oligonucleotides with a primary alkylamine group is a widely used method for introducing additional functional groups to the RNA. In particular, several 5'-modifications of RNA molecules such as sulfhydryl modification for functionalizing the 5'-terminus of RNA by a transcription or kinase reaction have been shown to have broad applications in studying RNA structures, mapping RNA-protein interactions, and in vitro selection of catalytic RNAs, since a unique functional group incorporated into the RNA can be subsequently conjugated to the desired molecule by a selective chemical reaction. However, there is still a need to develop coupling chemistry with high stability and yield to modify RNA and other biomolecules. In addition, the coupling functional groups are ideally required to be stable under aqueous reaction conditions, and the coupling reaction should be highly chemoselective. In this regard, Cu-catalyzed azide-alkyne cycloaddition (CuAAC or click chemistry) to form the triazole version of Huisgen’s [2+3] cycloaddition family may be the best choice, because this reaction only occurs between alkynyl and azido functional groups with high yield, and because the resulting 1,2,3-triazoles are stable at aqueous conditions and high temperature. Indeed, the azide group is one of the most utilized bioorthogonal chemical tags for biomolecule-conjugate experiments because of its small size and inertness to most components in a biological environment. We recently reported a two-step synthetic method for 5'azido-5'-deoxyguanosine (azido-G) by adapting literature procedures (Scheme 1) and its efficiency for click chemistry using 6-heptynoyl p-nitroaniline, in consideration that the click chemistry with rapid reaction between the azide and alkyne groups to form a covalent triazole linkage without cross reacting with other functional groups could be used in bioconjugation chemistry. In the present study, we sought enzymatic methods to incorporate azido groups in RNA and measured incorporation efficiency of azido-G into the 5'terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription. This is important in consideration that a terminal azido group is able to be introduced to the 5'termini of RNA molecules, and that azides are unstable under solid-phase synthesis conditions. A 97-mer single-stranded DNA containing a T7 promoter at the 3'-end (5'CAG GAC TGC TCT CAC TCT CAC GCA CCA AGA AGC TGC CAT TGA TCC CGC TGC

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