Synthesis, Derivatization and Photochemical Control of ortho‐Functionalized Tetrachlorinated Azobenzene‐Modified siRNAs

Abstract

We report the chemical synthesis and derivatization of an ortho‐functionalized tetrachlorinated azobenzene diol. A 4′,4‐dimethoxytrityl (DMT) phosphoramidite was synthesized for its site‐specific incorporation within the sense strand of an siRNA duplex to form ortho‐functionalized tetrachlorinated azobenzene‐containing siRNAs (Cl‐siRNAzos). Compared to a non‐halogenated azobenzene, ortho‐functionalized tetrachlorinated azobenzenes are capable of red‐shifting the π→π* transition from the ultraviolet (UV) portion of the electromagnetic spectrum into the visible range. Within this visible range, the azobenzene molecule can be reliably converted from trans to cis with red light (660 nm), and converted back to trans with violet wavelength light (410 nm) and/or thermal relaxation. We also report the gene‐silencing ability of these Cl‐siRNAzos in cell culture as well as their reversible control with visible light for up to 24 hours.

Controlling gene expression with light: a multidisciplinary endeavour

“…Attaching an anthracene functionality along with a porphyrin photosensitiser onto the termini of siRNA controlled its function with green or red light [ 67 ]. Hybridisation of the siRNA strands was also controlled through incorporation of an azobenzene into the backbone and controlled with red light [ 68 ]. 2-Nitrobenzyl-photocaging of the Watson–Crick face of a single nucleobase in siRNA inhibited siRNA:mRNA duplex formation, and thus knocking down a reporter protein [ 69 ] and an endogenous gene [ 70 ].…”

General Principles for the Design of Visible‐Light‐Responsive Photoswitches: Tetra‐ortho‐Chloro‐Azobenzenes

“…Te tra-ortho-substituted azobenzenes emerged as privileged light-responsive molecular photoswitches,w ith good absorption band separation and half-lives of the metastable cis isomer in the range that enables multiple applications. [27][28][29][34][35][36] Among them, azobenzenes with all four ortho positions substituted with chlorine atoms ( Figure 1A), have already enabled using green and even red light to control peptide conformation, [27] antibiotic potency, [37] ion channel activity, [38][39][40] and the function of nucleic acids [41] and ion receptors [42,43] for controlling the transport through biological membranes [44] ( Figure 1D). However,w hile for normal azobenzenes several relationships between structure and photochemical properties have been defined, only little systematic information is available for the tetra-ortho-substituted systems,making their design largely atrial-and-error endeavor.T he main difference between those switches and classical azobenzenes comes from the fact that, for their operation in both directions,v isible-light absorption bands are used, which correspond to weakly allowed transitions of np*c haracter ( Figure 1B,C).…”

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

“…These alterations to the benzene ring of the azobenzene put strain on the N=N bond and allows for lower energy wavelength light to be used instead of potentially toxic UV light. In our most recent publication, we could get the same results as in Figure 3, but without UV light, and without having to carefully monitor the dose since red light is not toxic, the wavelength is too high (Hammill, Islam, & Desaulniers, 2020b). While this was an excellent result, moving away from UV light, an unfortunate consequence of this modification was the half‐life of the cis conformer was reduced from 4 hr to 2 min at 37°C.…”

“…These unique and modular properties allow azobenzene to be used in many applications, including its incorporation into oligonucleotides (Hammill et. al., 2017;Hammill et al" 2018;Hammill & Desaulniers, 2020;Hammill et al, 2020aHammill et al, , 2020b.…”

“…the wavelength is too high (Hammill, Islam, & Desaulniers, 2020b). While this was an excellent result, moving away from UV light, an unfortunate consequence of this modification was the half-life of the cis conformer was reduced from 4 hr to 2 min at 37°C.…”