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

Abstract

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 reactivates 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.

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

“…The most critical application, which we demonstrated in several studies (Hammill et al., 2017; Hammill et al., 2018; Current Protocols article: Hammill et al., 2020a; Hammill et al., 2020b; 2020c), was the ability of the azobenzene to disrupt the duplex when exposed to UV light in such a way that we theorized it would prevent the loading of the duplex of an azobenzene‐containing siRNA into the RISC complex. We observed this very trend, and further theorized that visible light would allow RISC loading to resume, which is what was observed.…”

“…Equal amounts of sense and anti-sense stock must be combined and then annealed in binding buffer; see Basic Protocol 1 of Current Protocols article: Hammill et al, 2020a. 53. For each desired final concentration (for a 12-well plate, final volume: 1 ml) make a serial dilution from the stock solution so that adding 1 μl of Cl-siRNAzo will give the desired final concentration for a 1-ml final volume.…”

“…For example, it has been incorporated into peptides because it has a high quantum yield when photo‐switching (Beharry & Woolley, 2011). These unique and modular properties allow azobenzene to be used in many applications, including its incorporation into oligonucleotides (Hammill, Isaacs‐Trepanier, & Desaulniers, 2017; Hammill, Patel, Abd Alla, & Desaulniers, 2018; Current Protocols article: Hammill, Islam, & Desaulniers, 2020a; Hammill, Islam, & Desaulniers, 2020b, 2020c).…”

Photochemical modifications for DNA/RNA oligonucleotides

“…Newer applications include siRNAzos 151 in gene silencing in cells and in vivo : siRNAzos use AzoB as internal spacers within the sense strand in HeLa cells. 138 siRNAzos in the cis form would distort the siRNA helix, thus rendering it non-functional, but irradiation with UV light would make it functional and lead to gene silencing.…”

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

“…For example, it has been incorporated into peptides because it has a high quantum yield when photoswitching (Beharry & Woolley, 2011;Li et al, 2014). Other uses include the modulation of the nucleobase of an oligonucleotide via chemical modification, which makes azobenzene attractive for many applications, including oligonucleotide enhancement (Goldau et al, 2015;Hammill et al, 2017Hammill et al, , 2018Hammill et al, , 2020aHammill et al, , 2020bHammill et al, , 2020cHammill et al, , 2022Lubbe et al, 2017;Wu et al, 2015). However, despite its robustness, it does have limitations.…”