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

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Only one example of small molecule-induced activation of antisense function exist. This has been restricted to cyclic caged morpholinos that, based on sequence, can have significant background activity in the absence of the trigger. Here, we provide a new approach using 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, efficiently 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 frog embryos. We validated the general applicability of this approach by targeting three different genes. The incorporation of azidomethylene (Azm)-caged nucleobases into morpholino oligonucleotides via solid phase synthesis, enabled by the unique compatibility of azides with P(V) chemistry, allowed for conditional control of antisense function. The developed Azm-caged MOs (azMOs) were readily activated by a small molecule phosphine trigger through a Staudinger reduction and exhibited robust knockdown of exogenous and endogenous genes in cells and aquatic embryos. Furthermore, orthogonal knockdown of two genes in zebrafish embryos were enabled through use of azMO in combination with a photoactivatable MO.+ image