Photochemical control of DNA decoy function enables precise regulation of nuclear factor κB activity

Abstract

DNA decoys have been developed for the inhibition of transcriptional regulation of gene expression. However, the present methodology lacks the spatial and temporal control of gene expression that is commonly found in nature. Here, we report the application of photoremovable protecting groups on nucleobases of nuclear factor κB (NF-κB) DNA decoys to regulate NF-κB-driven transcription of secreted alkaline phosphatase using light as an external control element. The NF-κB family of proteins is comprised of important eukaryotic transcription factors that regulate a wide range of cellular processes and are involved in immune response, development, cellular growth, and cell death. Several diseases, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease, have been linked to constitutively active NF-κB. Through the direct incorporation of caging groups into an NF-κB decoy, we were able to disrupt DNA:DNA hybridization and inhibit the binding of the transcription factor to the DNA decoy until UV irradiation removed the caging groups and restored the activity of the oligonucleotide. Excellent light-switching behavior of transcriptional regulation was observed. This is the first example of a caged DNA decoy for the photochemical regulation of gene expression in mammalian cells and represents an important addition to the toolbox of light-controlled gene regulatory agents.

Figure 1

In vitro binding of NF-κB DNA decoys to the NF-κB protein complex. Nuclear extracts were isolated from NF-κB/SEAP HEK 293 cells and incubated with radiolabelled NF-κB DNA decoys at room temperature for 20 min. Samples were analyzed on a 16% native PAGE gel and imaged with a Typhoon 7000 phosphorimager.

Figure 2

Light-activation of NF-κB DNA decoy binding. Nuclear extracts were isolated from NF-κB/SEAP HEK 293 cells and caged decoys were irradiated for 1 min (365 nm, 25 W) and incubated with nuclear extracts at room temperature for 20 min. Samples were analyzed on a 16 % Native PAGE gel and imaged with a Typhoon 7000 phosphorimager.

Figure 3

Photochemical activation of NF-κB induced SEAP expression. NF-κB/SEAP HEK293 cells were transfected with caged and non-caged DNA decoys using X-tremeGENE. Cells were either irradiated for 2 min (365 nm, 25 W) or kept in the dark. TNFα was added after 4 hours, and a SEAP assay was conducted after 24 hours using a Phospha Light Systems kit (Applied Biosystems). Cell viability was assayed using a Cell Titer Glo assay (Promega), and the SEAP signal was normalized to the Cell Titer Glo signal. All experiments were performed in triplicate and error bars represent standard deviations.

Figure 4

Temporally controlled deactivation of SEAP expression with caged NF-κB decoys. NF-κB/SEAPorter HEK293 cells were transfected with inactive (D0), non-caged (D5), and caged (D6) DNA decoys using X-tremeGENE, followed by TNFα addition 4 hrs post-transfection. Aliquots of the supernatant were taken at 1, 8, 12, 24 and 48 hrs after induction with TNFα followed by SEAP quantification. Cells were irradiated for 2 min (365 nm, 25 W) at 12 hrs after induction. The SEAP signal (Phospha Light Systems kit, Applied Biosystems) was normalized to cell viability (Cell Titer Glo, Promega). All experiments were performed in triplicate and error bars represent standard deviations. Asterisks indicate t-test results: * p < 0.5, ** p < 0.05, and *** p < 0.0005.

Scheme 1

NF-κB regulated transcription in the presence of DNA decoys. A) Upon stimulation, NF-κB translocates into the nucleus and binds to its DNA binding site activating transcription. B) In the presence of DNA decoys, NF-κB binds to the DNA decoy over its genomic binding site, leading to an inhibition of transcription.

Scheme 2

Photochemical control over NF-κB activation of gene expression. The caging groups disrupt Watson-Crick base-pairing of DNA and thus hairpin formation, rendering the decoy inactive. Therefore, NF-κB binds to its native genomic binding site and initiates transcription. After a brief irradiation with UV light, the caging groups are removed, the hairpin decoy forms, and out-competes the natural NF-κB binding site, leading to the inhibition of gene expression.