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

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 (Tm ) 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.

Synthesis of Dinucleotide Non-Symmetrical Triester Phosphate Phosphoramidites and Their Incorporation Within Oligonucleotides.

In this protocol article, the synthesis of dinucleotide non-symmetrical triester phosphate phosphoramidites will be highlighted. Specifically, we use a selective transesterification starting with tris(2,2,2-trifluoroethyl) phosphate to afford a dinucleotide derivative phosphate ester. Substitution of the final trifluoroethyl group with various alcohols affords a dinucleotide triester phosphate with a hydrophobic group, which can then be deprotected and converted to a phosphoramidite for incorporation within oligonucleotides. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of a DMT- and TBS-protected unsymmetrical dinucleotide Basic Protocol 2: Synthesis of a DMT-protected unsymmetrical dinucleotide phosphotriester monoalcohols Basic Protocol 3: Synthesis of DMT-protected phenylethyl phosphotriester dinucleotide phosphoramidites Basic Protocol 4: Synthesis, purification, and characterization of RNAs containing triester phosphate modifications.

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

This paper contains the detailed synthesis and characterization protocols of ortho-functionalized tetrachlorinated azobenzene-containing small interfering RNAs (siRNAs), which have photoswitchable properties effectively controlled with visible light. To design this tetrachlorinated azobenzene scaffold, a late-stage chlorination with N-chlorosuccinimide and palladium is used. Next, a single hydroxyl group from the tetrachlorinated azobenzene is protected with a 4,4'-dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate tetrachlorinated azobenzene-containing oligonucleotides. This paper also contains the detailed biophysical characterization, biological testing, and photo-switching protocols of ortho-functionalized chlorinated azobenzene-containing siRNAs (Cl-siRNAzos), which have photoswitchable properties that can be controlled with visible light. First, the Cl-siRNAzos are characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (Tm ) of the duplexes. Secondly, the biological testing of the Cl-siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to red light in order to inactivate the Cl-siRNAzo, and then either violet light or infrared thermal relaxation is deployed, which re-activates the Cl-siRNAzo. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxyethyl) ortho-functionalized tetrachlorinated azobenzene phosphoramidite (5) Basic Protocol 2: Synthesis, purification, and characterization of siRNAs containing ortho-functionalized tetrachlorinated azobenzene Basic Protocol 3: Gene-silencing evaluation of ortho-functionalized tetrachlorinated azobenzene using firefly luciferase.

Synthesis, Derivatization and Photochemical Control of an ortho-Functionalized Tetrafluorinated Azobenzene-Modified siRNA.

We report the synthesis of an ortho-functionalized tetrafluorinated azobenzene phosphoramidite for its site-specific incorporation into RNA. The tetrafluorinated azobenzene is embedded within the antisense strand of an siRNA duplex to form an ortho-functionalized tetrafluorinated azobenzene-containing siRNA (F-siRNAzo). The F-siRNAzo is inactivated via trans to cis conversion with green light (530 nm), and reactivated with blue light (470 nm) via cis to trans conversion in cell culture. The long half-life and stability of the tetrafluorinated azobenzene unit allows for reversible control of the F-siRNAzo in cell culture for up 72 hours.

SiRNAs with Neutral Phosphate Triester Hydrocarbon Tails Exhibit Carrier-Free Gene-Silencing Activity.

Short interfering RNAs (siRNAs) show promise as gene-silencing therapeutics, but their cellular uptake remains a challenge. We have recently shown the synthesis of siRNAs bearing a single neutral phenylethyl phosphotriester linkage within the sense strand. Here, we report the synthesis of siRNAs bearing three different hydrophobic phosphate triester linkages at key positions within the sense strand and assess their gene silencing in the absence of a transfection carrier. The best siRNAs bearing hydrophobic phosphate triester tails were not aromatic and exhibited effective gene silencing (IC50 ≈ 56-141 nM), whereas the aromatic derivative with three hydrophobic tails did not exhibit carrier-free gene silencing.

Building siRNAs with Cubes: Synthesis and Evaluation of Cubane-Modified siRNAs.

Cubane molecules hold great potential for medicinal chemistry applications due to their inherent stability and low toxicity. In this study, we report the synthesis of a cubane derivative phosphoramidite for the incorporation of cubane into small interfering RNAs (siRNAs). Synthetic siRNAs rely on chemical modifications to improve their pharmacokinetic profiles. However, they are still able to mediate sequence-specific gene silencing via the endogenous RNA interference pathway. We designed a library of siRNAs bearing cubane at different positions within the sense and antisense strands. All siRNAs showed excellent gene-silencing activity, with IC50 values ranging from 45.4 to 305 pM. Incorporating the cubane modification in both the sense and antisense strand led to viable duplexes with good biological activity. To the best of our knowledge, this is the first report of siRNAs bearing a cubane derivative within the backbone.

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 (Tm ) 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).

Synthesis and Evaluation of Neutral Phosphate Triester Backbone-Modified siRNAs.

Two unsymmetrical dinucleotide phosphate triesters were synthesized via transesterification from tris(2,2,2-trifluoroethyl) 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 gene-silencing ability, siRNA strand selection, and resistance to nucleases. These backbone-modified phosphate triester siRNAs offer many improvements compared to natural unmodified siRNAs.

Synthesis of Azobenzene Derivative Phosphoramidites for Incorporation into Oligonucleotides.

This article contains the detailed synthesis and characterization protocols of azobenzene containing siRNAs, which have photoswitchable properties effectively controlled with light. First, the azobenzene scaffolds are synthesized via reduction of nitrophenyl alcohols in the presence of zinc. Next, the hydroxyl group of azobenzene derivatives are protected with a dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate azobenzene-containing oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxymethyl)-azobenzene phosphoramidite Basic Protocol 2: Synthesis of 4,4'-bis(hydroxyethyl)-azobenzene phosphoramidite Basic Protocol 3: Synthesis, purification and characterization of oligonucleotides containing azobenzene derivatives.

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

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.

Effects of Chemical Modifications on siRNA Strand Selection in Mammalian Cells.

Small interfering RNAs (siRNAs) enable efficient gene silencing through RNA interference (RNAi) mechanisms. The RNAi machinery relies on an RNA-guided nuclease, Argonaute-2 (Ago2), which preferentially selects a single strand from an siRNA duplex. Complementarity between the selected strand and an RNA target strand leads to silencing through cleavage. The U.S. Food and Drug Administration's recent approval of two siRNA drugs has reignited optimism for RNAi therapeutics. Despite this recent success in the field, off-target effects are still a major concern; however, chemical modifications have shown promise in mitigating some off-target gene silencing. To evaluate the impact of novel chemical modifications on strand selection, we developed a quantitative polymerase chain reaction-based assay that is compatible with several pre-existing siRNA libraries and was used to characterize chemically modified siRNAs. siRNAs bearing azobenzene and propargyl modifications at the central region of the passenger strand significantly improved strand selection. On the other hand, folic acid-modified siRNAs improved strand selection best when placed at the 3' terminus. This study highlights the development and utility of a convenient method to evaluate the impact that novel chemical modifications have on strand-specific gene silencing of siRNAs.

Reversible control of RNA interference by siRNAzos.

In this study, we report the reversible control of RNA interference using siRNAzos, a class of siRNAs that contain azobenzene. Herein, we demonstrate that it is possible to take an active siRNAzo, and inactivate it for up to 24 hours. We also demonstrate reversibility of these siRNAzos within cell culture. For example, active siRNAzos can be inactivated in cell culture with ultraviolet light, and then reactivated with visible light. In addition, we also show that siRNAzos can be activated and inactivated towards the endogenous target gene, BCL2.

Stability and evaluation of siRNAs labeled at the sense strand with a 3'-azobenzene unit.

siRNAs bearing a 3'-azobenzene derivative on the sense strand were evaluated for their gene silencing ability in mammalian cell culture and nuclease stability in nuclease-rich media. Azobenzene can be isomerized between cis and trans isomers through the incubation of UV (cis isomer) and visible light (trans isomer). It was demonstrated that subtle differences in nuclease stability and activity were observed. These small changes can be used to photochemically fine-tune the activity of an siRNA for gene-silencing applications.