Optochemical control of RNA interference in mammalian cells

Abstract

Short interfering RNAs (siRNAs) and microRNAs (miRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules.

Scheme 1.

Two siRNA light-activation approaches. Caged nucleotides are positioned (A) near the cleavage site or (B) at the seed region of the siRNA agent, leading to gene expression by preventing RISC cleavage or mRNA target recognition. On UV irradiation, the caging groups are cleaved resulting in the silencing of gene expression through RNA interference.

Scheme 2.

Synthesis of the NPOM-caged guanosine phosphoramidite 7 from commercially available 1. Dimethylformamide (DMF), tert-butyldimethylsilyl chloride (TBDMSCl), dichloromethane (DCM), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), di(p-methoxyphenyl)phenyl-methyl chloride (DMTCl) and tetrahydrofuran (THF).

Scheme 3.

Synthesis of the NPOM-caged uridine phosphoramidite 13 from 8. Dimethylformamide (DMF), tert-butyldimethylsilyl chloride (TBDMSCl), dichloromethane (DCM), di(p-methoxyphenyl)phenyl-methyl chloride (DMTCl), tetrahydrofuran (THF) and N,N-diisopropylethylamine (DIPEA).

Figure 1.

Sequences and T_m of caged siRNAs. Bold and underLined G denotes a caged guanosine nucleotide from the incorporation of 7 and a bold and underLined U denotes a caged uridine nucleotide from the incorporation of 13. Standard deviations were calculated form three individual experiments.

Figure 2.

Photoactivated RNA interference in mammalian cells. HEK 293T cells were transfected with pEGFP-N1, pDsRed-N1 monomer and siRNA oligonucleotides. Cells were irradiated for 5 min (25 W, 365 nm) or kept in the dark. (A–F) Cells were imaged after 48 h. The GFP channel is shown above the DsRed channel. (G) After a 48-h incubation, the cells were trypsinized and analyzed by flow cytometry. The number of cells expressing both GFP and DsRed was normalized to the number of cells expressing only DsRed. Standard deviations were calculated from three individual experiments.

Figure 3.

Optochemical activation of Eg5 siRNA in HeLa cells. HeLa cells were transfected with caged and non-caged siRNAs (40 pmol). The cells were irradiated (5 min, 25 W, 365 nm) and incubated at 37°C, 5% CO₂ for 48 h. (A–G) The cells were fixed and stained with Alexa Fluor 488 phalloidin (green) and DAPI (blue). The cells were imaged on a Zeiss LSM 710 confocal microscope using a 40× oil objective and Alexa Fluor 488 and DAPI-specific lasers (488 nm multiline argon and 405 nm diode).

Figure 4.

Quantification of photoactivation of Eg5 siRNA. HeLa cells were transfected with siRNAs, and after 48-h incubation, the RNA was extracted and quantitative real-time PCR analysis was performed. Eg5 expression was normalized to the expression of the GAPDH housekeeping gene, and the negative control was set to 100% Eg5 expression. Error bars represent standard deviations from three independent experiments.