Tetrazine-Induced Bioorthogonal Activation of Vitamin E-Modified siRNA for Gene Silencing

Abstract

The temporal activation of siRNA provides a valuable strategy for the regulation of siRNA activity and conditional gene silencing. The bioorthogonal bond-cleavage reaction of benzonorbonadiene and tetrazine is a promising trigger in siRNA temporal activation. Here, we developed a new method for the bio-orthogonal chemical activation of siRNA based on the tetrazine-induced bond-cleavage reaction. Small-molecule activatable caged siRNAs were developed with the 5′-vitamin E-benzonobonadiene-modified antisense strand targeting the green fluorescent protein (GFP) gene and the mitotic kinesin-5 (Eg5) gene. The addition of tetrazine triggered the reaction with benzonobonadiene linker and induced the linker cleavage to release the active siRNA. Additionally, the conditional gene silencing of both exogenous GFP and endogenous Eg5 genes was successfully achieved with 5′-vitamin E-benzonobonadiene-caged siRNAs, which provides a new uncaging strategy with small molecules.

Lipid and Peptide-Oligonucleotide Conjugates for Therapeutic Purposes: From Simple Hybrids to Complex Multifunctional Assemblies

“…The best option in terms of higher loading and efficiency was the conjugation of vitamin E (Sheme 1) [ 83 ]. Recently, siRNAs were modified with vitamin E by a benzonorbonadiene linker, which releases active siRNAs when reacting with tetrazines [ 84 ].…”

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

“…In addition to multiple model systems, general applicability of the approach was demonstrated by targeting three different genes ( EGFP , ntla , and bmp4 ) requiring MO sequences that differ in the positions of the Azm group installations and in G‐content. Development of conditionally controlled gene knockdown in aquatic embryos is important because previously reported small molecule regulation of RNAi activity in cells [14b,c] is not translatable due to the general inefficiency of RNAi in teleosts [15] . Enabled by our rapid azMOs activation, we demonstrated temporal control over the ntla silencing in zebrafish at different time points, supporting observations of its native expression pattern during development [49] .…”

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

“…To date, there are very few examples of small molecule‐triggered activation of gene silencing agents [14] . There are two reports of small molecule control of RNA interference (RNAi) in mammalian cells through use of a trans ‐cyclooctene (TCO)‐ and norbornene‐caged antisense strand that prevent formation of the RNAi silencing complex (RISC) until being activated by a tetrazine [14b,c] . While RNAi presents an effective gene knockdown approach in mammalian cells, it suffers from impaired target degradation in teleosts due to a mutated Argonaute‐2 (Ago‐2) protein, and is associated with major off‐target effects [15] .…”