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.

Double-Lariat Caged Morpholino Oligonucleotides for Optical Gene Silencing

“…18,23 In this case, the DMNB moiety was strategically linked with a masked hydroxymethylaniline scaffold through a carbamate linkage, which was also employed in our nitroreductase-and small molecule-activatable cMOs. 28,29 The spontaneous 1,6-elimination reaction has been reported to be rapid (t1/2 ~ 90 s), 36 enabling cMO activation to occur within minutes after linker photocleavage. The methylenetriazole moiety in the benzylic position is further expected to accelerate the reaction.…”

“…In addition, the modular linker chemistry of cyclic cMOs facilitates their design for other uncaging methods. For instance, while the first generation of cyclic cMOs was designed to be activated by ultraviolet light, we have subsequently reported cyclic cMOs that can be uncaged with blue light, 26,27 enzymes, 28 and small molecules 29 . Other versions of cyclic cMOs that contain ruthenium-or BHQ-based linkers have also been reported.…”

Optical control of CRISPR-Cas editing with cyclically caged guide RNAs

“…3D), which again proven that the head-to-tail closure may be a generic strategy for the modulation of gRNA function in gene editing. 64-65 We further investigated whether a rapid exposure with UV light would restore the Cpf1 cleavage.…”

“…3D), which again proven that the head-to-tail closure may be a generic strategy for the modulation of gRNA function in gene editing. [64][65] We further investigated whether a rapid exposure with UV light would restore the Cpf1 cleavage. Specifically, a 60 seconds' irradiation evidently activated the Cpf1 function by fully releasing the parent linear GFP-Cpf1-sgRNA (Fig.…”

Ortho-Phosphinoarenesulfonamide-Mediated Staudinger Reduction of Aryl and Alkyl Azides

“…Staudinger reduction [ 1 , 2 , 3 ] is one of the most common transformations used to prepare amino compounds in organic synthesis. Recently, the biorthogonal property [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ] of organic azides [ 12 ] has significantly enhanced the visibility of the Staudinger reaction in the context of chemical biology and medicinal chemistry. The classic Staudinger protocol employs trivalent phosphines, predominately triphenylphosphine, to mediate the reduction of organic azides under aqueous conditions via an aza -ylide (or iminophosphorane) intermediate [ 13 , 14 ] ( Scheme 1 A).…”