Controlling Gene‐Silencing with Azobenzene‐Containing siRNAs (siRNAzos)

This article contains the detailed biophysical characterization, biological testing, and photo‐switching protocols of azobenzene containing siRNAs (siRNAzos), which have photoswitchable properties that can be controlled with light. First, the siRNAzos are characterized by annealing the sense and anti‐sense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures ( T m ) of the duplexes. Second, the biological testing of the siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene‐silencing activities are measured after exposure to ultraviolet (UV) light in order to inactivate the siRNAzo, and then broadband visible light, which re‐activates the siRNAzo. This inactivation/reactivation protocol can be done in real time, and is reversible and robust and can be performed multiple times on the same sample if desired. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Bio‐physical characterization of siRNAzo duplexes Basic Protocol 2 : Evaluation of azobenzene gene‐silencing using Firefly Luciferase Basic Protocol 3 : Evaluation of azobenzene gene‐silencing using reverse transcriptase‐polymerase chain reaction (RT‐PCR)