Incorporation Efficiency of 5'-Azido-5'-Deoxyguanosine into 5'-Terminus of RNA for Preparation of Azido-Functionalized RNA

摘要

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 derivati-zation 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 function-alizing 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:-cata-lyzed 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.