Genetically encoded light-activated transcription for spatiotemporal control of gene expression and gene silencing in mammalian cells

Abstract

Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the site-specific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.

Figure 1. The light-activated T7RNAp is expressed in mammalian cells through site-specific incorporation of a photocaged lysine (PCK) into its active site in response to an amber stop codon (TAG) via an engineered tRNA synthetase (PCKRS) that misacylates an amber-suppressor tRNA (PylT) with PCK.

The caged T7RNAp is completely inactive until irradiation with 365 nm UV light induces decaging and activation of T7-driven transcription. Depending on the function of the transcribed RNA, light-induced protein expression from mRNA or light-induced gene silencing via RNA interference from shRNA is achieved.

Figure 2. Structural representation of K631-caged (left) and wild-type (right) T7RNAp.

The K631 residue is indicated in green, while the DNA template and RNA transcript are indicated in red and yellow, respectively. The photocaged lysine is predicted to block transcription until UV irradiation removes the caging group and restores T7RNAp activity. Images were generated in PyMol from PDB 1S76.

Figure 3. Expression of caged T7RNAp and photochemical activation.

A) Mammalian cells were transfected with expression plasmids for the PylT, PCKRS, and T7RNApK631TAG and grown in the presence of PCK (2 mM) for 24 hours. Western blot analysis was performed using a 6-His primary antibody with a FITC secondary antibody for detection, showing PCK dependent expression. B) HEK293T cells were transfected with plasmids expressing the PylT, PCKRS, T7RNApK631TAG, and T7-IRES-Luc were incubated with PCK overnight. UV irradiations were performed on a 365 nm transilluminator at increasing intervals. A luciferase bright glow assay was performed 24 hours after UV exposure. Luminescence units were normalized to the activation observed after a 20 minute irradiation and error bars represent the standard deviation obtained from experimental triplicates.

Figure 4. Light-activation of EGFP transcription in HEK293T cells.

A) Cells were co-transfected with the PCKRS, PylT, T7RNApK631TAG and T7-IRES-EGFP constructs then incubated with 2 mM PCK for 24 hours. Irradiation was then performed for 2 minutes at 365 nm, and cells were imaged 48 hours after UV exposure. Scale bars indicate 200 μm. B) Fluorescent cell counting of caged T7RNAp-driven EGFP expression of cells 48 hours after UV exposure. The frequencies of EGFP-positive cells (gated/total) were normalized to the 10 minute UV irradiation and error bars represent the standard deviation obtained from three experimental replicates.

Figure 5. Spatial control of photochemically activated gene expression.

Cells were transfected as previously described with the EGFP reporter for T7RNAp activity. Masks were used to irradiate a small population of cells at 365 nm for 2 minutes, and EGFP expression was observed at 48 hours. Irradiations were performed through a small pin hole (A) and a horizontal slit (B). Scale bars indicate 200 μm. Only the cells within the irradiated region express EGFP, demonstrating spatial control of K631-caged T7RNAp in mammalian cells.

Figure 6. Light-activation of Eg5 shRNA using the caged T7RNAp system, followed by RNA interference and triggering of binucleated cell phenotype.

Cells were transfected with an Eg5 siRNA (50 pmol) and the K631-caged T7RNAp expression system as previously described, followed by fixing and staining with DAPI (nucleus) and Rho-phalloidin (actin) prior to imaging. Scale bars indicate 20 μm and binucleated cells are labeled with a white arrow. A binucleated phenotype was observed with both the Eg5 siRNA and plasmid-based shRNA controls, while EGFP expression successfully tracked cells for the identification of T7RNAp-driven shRNA activity. The shRNA construct has been validated to enable light-activated RNA interference. The EGFP expression and cellular binucleation were fully dependent upon UV irradiation and activation of the caged T7RNAp.