Activation and deactivation of DNAzyme and antisense function with light for the photochemical regulation of gene expression in mammalian cells

Abstract

The photochemical regulation of biological systems represents a very precise means of achieving high-resolution control over gene expression in both a spatial and a temporal fashion. DNAzymes are enzymatically active deoxyoligonucleotides that enable the site-specific cleavage of RNA and have been used in a variety of in vitro applications. We have previously reported the photochemical activation of DNAzymes and antisense agents through the preparation of a caged DNA phosphoramidite and its site-specific incorporation into oligonucleotides. The presence of the caging group disrupts either DNA:RNA hybridization or catalytic activity until removed via a brief irradiation with UV light. Here, we are expanding this concept by investigating the photochemical deactivation of DNAzymes and antisense agents. Moreover, we report the application of light-activated and light-deactivated antisense agents to the regulation of gene function in mammalian cells. This represents the first example of gene silencing antisense agents that can be turned on and turned off in mammalian tissue culture.

Figure 1

Caging and decaging of DNA. A caged monomeric building block (here, thymidine phosphoramidite) is incorporated into a deoxyoligonucleotide through standard DNA synthesis, rendering the oligomer inactive. Upon a brief irradiation with UV light, the caging group is removed, restoring the natural thymidine residue and thus the biological function of the DNA (e.g. the ability to undergo duplex formation, or catalytic activity). The 6-nitropiperonyloxymethylene (NPOM) caging group is shown in blue.

Figure 2

Structures of caged DNA nucleosides employed in the photochemical regulation of DNA function. The light-removable caging groups are shown in blue.

Figure 3

Light-activated DNAzyme. The caged 10-23 DNAzyme D2 (40 nM) binds to its complementary RNA (4 nM), but has no catalytic activity due to incorporation of the caged thymidine 3 at the crucial position T₁₂ in the catalytic core. Irradiation at 365 nm removes the caging group, activates the DNAzyme, and induces RNA cleavage at a Mg²⁺ concentration of 100 mM (15 mM Tris buffer, pH 8.2). The RNA degradation was imaged by gel-separation of a ³²P labeled RNA substrate.

Figure 4

Time course of RNA cleavage by the DNAzymes D1 (non-caged; 40 nM) and D2 (caged; 40 nM) under different irradiation and re-folding conditions. All reactions were performed with 4 nM ³²P-labeled RNA substrate (10 mM MgCl₂, pH 8.2, 15 mM Tris buffer). RNA cleavage was assessed via the removal of aliquots of the sample at given time points, followed by PAGE analysis (see Figure 3) and quantification of the radioactively labeled RNA substrate with ImageQuant. All experiments were conducted in triplicate and the error bars represent standard deviations.

Figure 5

Photochemical deactivation of the DNAzyme D1 (40 nM) with the caged complementary DNA decoy DD2 (40 nM) in the presence of RNA substrate (4 nM; 10 mM MgCl₂, pH 8.2, 15 mM Tris buffer). Prior to irradiation the decoy is inactive and does not undergo hybridization to the DNAzyme; however, upon a brief irradiation (1 min, 365 nm, 25 W), the caging groups are removed enabling the hybridization of the decoy to the DNAzyme and inhibiting RNA cleavage.

Figure 6

Photochemical DNAzyme inactivation using the caged DNA decoy DD4 (4 nM) complementary to the catalytic core of the DNAzyme (40 nM) in the presence of RNA substrate (4 nM; 10 mM MgCl₂, pH 8.2, 15 mM Tris buffer). Prior to UV irradiation normal DNAzyme function is observed. However, upon decaging (1 min, 365 nm, 25 W), the DNA decoy is capable of hybridizing to the catalytic core, completely inhibiting DNAzyme catalyzed cleavage of RNA.

Figure 7

Photochemical DNAzyme inactivation using a caged hairpin strategy. Prior to UV irradiation normal DNAzyme function of HP3 (40 nM) is observed. However, upon decaging (1 min, 365 nm, 25 W), the complementary sequence is capable of hairpin formation, thus disrupting RNA-binding and the catalytic core and thereby inhibiting DNAzyme HP1 catalyzed cleavage of RNA substrate (4 nM; 10mM MgCl₂, pH 8.2, 15 mM Tris buffer).

Figure 8

Terminal hairpins introduced on the DNAzyme R1 increase intracellular stability in mammalian tissue culture and allow for mRNA cleavage. Fluorescence image of HEK293T cells co-transfected with DsRed and GFP expressing plasmids and the DNAzymes R1 (non-caged) and R2 (caged at T₃₇). A) Transfection of the non-caged DNAzyme R1 leading to the silencing of DsRed expression. B) Transfection of the DNAzyme R2 caged at the essential residue T₃₇ in the catalytic core, previously shown to abrogate DNAzyme activity; however, in this case DNAzyme complete silencing of DsRed is still observed. C) Control DNAzyme R7 transfection leading to the expression of both DsRed and GFP. Scale bar = 200 μm.

Figure 9

Photochemical activation of antisense oligonucleotide function in mammalian tissue culture. A) Normal DsRed expression is observed prior to irradiation with light, since the caging groups prevent mRNA:DNA hybridization. B) Irradiated cells (365 nm, 2 min, 25 W) no longer express DsRed due to light-activation of the DNA leading to DsRed silencing. GFP/DsRed (left) corresponds to an overlay of the GFP and DsRed images, while only the DsRed channel is shown on the right. Scale bar = 150 μm.

Figure 10

Deactivation of antisense oligonucleotide function in mammalian tissue culture. A) The hairpin antisense agent R6 is active in the absence of light irradiation, silencing the DsRed gene. B) Irradiation of cells removes the caging groups, enabling the formation of a hairpin that blocks the antisense agent from recognizing the mRNA transcript. Inactivation of the antisense agent then allows for DsRed expression. GFP/DsRed (left) corresponds to an overlay of the GFP and DsRed images, while only the DsRed channel is shown on the right. Scale bar = 200 μm.

Figure 11

Measurement of DsRed and GFP fluorescence to quantify intracellular antisense activity. Transfections were performed as previously described, and the cells were either irradiated (2 min, 365 nm, 25 W), or not exposed to UV irradiation. After 48 hours fluorescence was measured on a Molecular Devices Gemini EM microplate spectrofluorimeter. Relative fluorescence (DsRed/GFP) normalized to a transfection in the absence of DNA is shown. Error bars represent standard deviations from three independent experiments. The mRNA translational suppression of ~30% with the non-caged analog R1 is in accordance with previous reports.