Post-synthetic chemical modification of RNA at the 2'-position of the ribose ring via "click" chemistry

Abstract

This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128). The invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.

Description

BACKGROUND OF THE INVENTION[0001]RNA interference (RNAi) is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner. The RNAi machinery can be harnessed to destruct any mRNA of a known sequence. This allows for suppression (knock-down) of any gene from which it was generated and consequently preventing the synthesis of the target protein. Smaller siRNA duplexes introduced exogenously were found to be equally effective triggers of RNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Bartel, D. P. Cell 2000, 101, 25-33). Synthetic RNA duplexes can be used to modulate therapeutically relevant biochemical pathways, including ones which are not accessible through traditional small molecule control.[0002]Chemical modification of RNA leads to improved physical and biological properties such as nuclease stability (Damha et al Drug Discovery Today ...

AI Technical Summary

Benefits of technology

[0015]This invention relates to the post-synthetic chemical modification of RNA at the 2′-position on the ribose ring via a copper catalyzed Huisgen cycloaddition (“click” chemistry: Kolb, Sharpless Drug Discovery Today 2003, 8, 1128). The invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.

Problems solved by technology

Chemical modifications of RNA have relied heavily on work-intensive, cumbersome, multi-step syntheses of structurally novel nucleoside analogues and their corresponding phosphoramidites prior to RNA assembly.

Furthermore, some chemical modifications bear sensitive functional groups that may be incompatible with state-of-the-art automated synthesis of RNA as well as subsequent downstream cleavage-deprotection steps.

These attributes have made chemical modification of RNA prior to synthesis rather low-throughput and limited in scope.

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