Synthesis of an Ortho‐Functionalized Tetrafluorinated Azobenzene Phosphoramidite for Incorporation into a Tetrafluorinated Azobenzene‐Containing siRNA for Photocontrolled Gene Silencing

Abstract This article presents the detailed synthesis and characterization protocols for an ortho‐ functionalized tetrafluorinated azobenzene containing siRNA, which has photoswitchable properties. To design this tetrafluorinated azobenzene scaffold, several synthetic steps are performed to generate a symmetrical tetrafluorinated azobenzene diol. This diol is treated with dimethoxytrityl chloride (DMT‐Cl) to protect one of the alcohols. Next, the DMT‐protected tetrafluorinated monoalcohol is phosphitylated to afford the DMT‐phosphoramidite building block used for solid‐phase synthesis. This paper also contains the detailed biophysical characterization, biological testing, and photo‐switching protocols of an ortho‐functionalized fluorinated azobenzene containing siRNA (F‐siRNA), which has photoswitchable properties that can be controlled with visible light. First, the F‐siRNA was characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile and melting temperature ( T m ) of the duplexes. Second, biological testing of the F‐siRNA is performed in cell culture to determine their gene silencing efficacy. Finally, their gene‐silencing activities are measured after exposure to green light to inactivate the F‐siRNA, followed by blue light, which reactivates the F‐siRNA. The F‐siRNA can be kept inactive for up to 72 hr and reactivated at any time within this 72‐hr window. © 2023 Wiley Periodicals LLC.