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.

Fine‐tuning the activity of short‐interfering RNAs (siRNAs) with light could help overcome several obstacles related to potency, delivery, and off‐target effects. In this study, we chemically modify siRNAs that contain azobenzene derivative spacers within the sense strand. These molecules are called siRNAzos and they are successfully accommodated within the RNAi pathway as measured by gene‐silencing dose‐dependent knockdown. In addition to its RNAi biocompatibility, we are able to photochemically control the activity of the siRNAzos that contain the azobenzene within the central region of the sense strand. We demonstrate it is possible to both inactivate and reactivate several siRNAzos with ultraviolet and visible light, respectively.

In this study, we report the reversible control of RNA interference using siRNAzos, a class of siRNAs that contain azobenzene.

Two unsymmetrical dinucleotide phosphate triesters were synthesized via transesterification from tris(2,2,2trifluoroethyl) phosphate. The protected triesters were phosphytilated to generate phosphoramidites for solid-phase oligonucleotide synthesis. Neutral phenylethyl phosphate-modified short-interfering RNAs (siRNAs) were synthesized and evaluated for their genesilencing ability, siRNA strand selection, and resistance to nucleases. These backbone-modified phosphate triester siRNAs offer many improvements compared to natural unmodified siRNAs.