Small Molecule Control of Morpholino Antisense Oligonucleotide Function through Staudinger Reduction

Abstract

Conditionally activated, caged morpholino antisense agents (cMOs) are tools that enable the temporal and spatial investigation of gene expression, regulation, and function during embryonic development. Cyclic MOs are conformationally gated oligonucleotide analogs that do not block gene expression until they are linearized through the application of an external trigger, such as light or enzyme activity. Here, we describe the first examples of small molecule-responsive cMOs, which undergo rapid and efficient decaging via a Staudinger reduction. This is enabled by a highly flexible linker design that offers opportunities for the installation of chemically activated, self-immolative motifs. We synthesized cyclic cMOs against two distinct, developmentally relevant genes and demonstrated phosphine-triggered knockdown of gene expression in zebrafish embryos. This represents the first report of a small molecule-triggered antisense agent for gene knockdown, adding another bioorthogonal entry to the growing arsenal of gene knockdown tools.

Figure 1.

Following exposure to a phosphine trigger, the p-azidobenzyl linker undergoes a Staudinger reduction, forming a p-aminobenzyl intermediate. The linker then rapidly self-cleaves via a 1,6-elimination and subsequent decarboxylation to generate the active, linearized MO, which can then hybridize to target mRNAs and silence gene expression.

Figure 2.

(A) Synthesis of p-azidobenzyl MO linker 9. (B) Application of the linker in the synthesis of the cMO targeting ntla.

Figure 3.

(A) The Q-rhodamine fluorophore is caged with p-azidobenzyl carbamates. Following treatment with phosphine, fluorescence is activated via Staudinger reduction-induced decaging. (B) Structures of phosphines. (C) The fluorescence activation of the sensor (5 μM) was monitored over time following incubation with various phosphines (50 μM) at 29 °C. Error bars represent standard deviations from the average of three independent experiments.

Figure 4.

(A) Representative images of zebrafish embryos injected with 10b and soaked in E3 water supplemented with 2DPBM (100 μM) for various durations. (B) Quantification of rhodamine fluorescence (n = 5 embryos/condition) demonstrates efficient activation of 10b following phosphine treatment and supports the micrograph findings. Error bars represent standard deviations from the average of 5 embryos.

Figure 5.

(A) In the presence of the cyclic p-azidobenzyl ntla cMO, luciferase is expressed, as cyclization impedes target binding and masks antisense activity. Treatment with phosphine induces cMO cleavage, generating the active, linearized MO which represses luciferase expression. (B) Titration of linear ntla MO induces dose-dependent silencing of Fluc expression. (C) Nearly complete silencing of Fluc expression is observed following 2DPBM-mediated activation of the cyclic p-azidobenzyl ntla cMO, while no effect on reporter expression is observed with cMO alone. Data represent the means ± standard deviation from at least three independent experiments.

Figure 6.

(A) Representative images of ntla morphant phenotypes in zebrafish embryos at 24 hpf and phenotypic scoring of embryos injected with the indicated morpholino reagent and soaked in E3 water supplemented with DMSO (0.2%) or 2DPBM (100 μM). (B) Representative images of spt morphant phenotypes in zebrafish embryos at 24 hpf and phenotypic scoring of zebrafish embryos injected with the indicated morpholino reagents and soaked in E3 water supplemented with DMSO (0.2%) or 2DPBM (100 μM). The scale bar equals 1 mm.