A chemical method for site-specific modification of RNA: The convertible nucleoside approach

Knowledge of RNA structure can greatly facilitate the understanding of its biological function. However, the physical properties of RNA, especially its conformational heterogeneity, present an impediment to high-resolution structural analysis. Thus, lower resolution methods such as biochemical probing, phylogenetic analysis, and molecular modeling have come to serve an important role in RNA science. This situation has created the need for a means by which to constrain RNA structure, either to reduce its conformational flexibility or to help distinguish between alternative structural models. To address this need, we have developed chemistry that permits the site-specific introduction of functionalizable tethers into RNA. Here we report the design and synthesis of reagents for use in solid-phase RNA synthesis that allow the functionalization of the base moiety of G, C, and A residues. Upon incorporation into oligonucleotides and subsequent treatment with alkylamines, the convertible nucleoside derivatives reported here give rise to functionally tethered N-4-alkyl-C, N-6-alkyl-A, and N-2-alkyl-G residues in RNA. The derivatized RNAs can then be used to target the attachment of chemical probes or the placement of disulfide crosslinks as structural constraints. The attachment of nonnatural functional groups to RNA in this fashion provides a powerful means of both probing its structural environment and constraining its conformation. The size and functionality of the N-alkyl modification is determined solely by the choice of alkylamine, thereby permitting the preparation of a wide range of functionally tethered RNAs.