Synthesis of Ortho-Functionalized Tetrachlorinated Azobenzene Phosphoramidites for Incorporation Into siRNAzos for Photocontrolled Gene Silencing.

This paper contains the detailed synthesis and characterization protocols of ortho-functionalized tetrachlorinated azobenzene-containing small interfering RNAs (siRNAs), which have photoswitchable properties effectively controlled with visible light. To design this tetrachlorinated azobenzene scaffold, a late-stage chlorination with N-chlorosuccinimide and palladium is used. Next, a single hydroxyl group from the tetrachlorinated azobenzene is protected with a 4,4'-dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate tetrachlorinated azobenzene-containing oligonucleotides. This paper also contains the detailed biophysical characterization, biological testing, and photo-switching protocols of ortho-functionalized chlorinated azobenzene-containing siRNAs (Cl-siRNAzos), which have photoswitchable properties that can be controlled with visible light. First, the Cl-siRNAzos are characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (T ) of the duplexes. Secondly, the biological testing of the Cl-siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to red light in order to inactivate the Cl-siRNAzo, and then either violet light or infrared thermal relaxation is deployed, which re-activates the Cl-siRNAzo. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxyethyl) ortho-functionalized tetrachlorinated azobenzene phosphoramidite (5) Basic Protocol 2: Synthesis, purification, and characterization of siRNAs containing ortho-functionalized tetrachlorinated azobenzene Basic Protocol 3: Gene-silencing evaluation of ortho-functionalized tetrachlorinated azobenzene using firefly luciferase.