Direct Activation of Nucleobases with Small Molecules for the Conditional Control of Antisense Function

Abstract

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Few examples of small molecule‐induced activation of antisense function exist, and have been restricted to cyclic morpholinos, which can have significant background activity in the absence of the trigger. Here, we provide a new approach by introducing azido‐caged nucleobases that are site‐specifically introduced into antisense morpholinos. The caging group design is a simple azidomethylene (Azm) group that, despite its very small size, blocks Watson‐Crick base pairing in a programmable fashion. Furthermore, it undergoes facile decaging via Staudinger reduction when exposed to a small molecule phosphine, generating the native antisense oligonucleotide under conditions compatible with biological environments. We demonstrated small molecule‐induced gene knockdown in mammalian cells, zebrafish embryos, and Xenopus embryos. We validate the general applicability of this approach by targeting three different genes.