Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro‐ and Eukaryotic Cells

Abstract

A light-activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site-specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradiation with nonphototoxic UV light of 365 nm removes the ortho-nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro- and eukaryotic systems allowed for the photochemical activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradiation of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradiation with UV light led to the activation of T7RNAP, thereby inducing RNA polymerization and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

“…The activity and photochemical control of K631-caged T7RNAp was investigated using a firefly luciferase reporter gene under control of the T7 promoter (Supporting Figure 1B). 39 First, the effects of UV exposure on the luciferase assay were studied, revealing that an irradiation time of 2 minutes and even up to 10-20 minutes had only small effects on T7-driven luciferase activity in cells expressing the wildtype polymerase (Supporting Figure 2). Light-activated luciferase expression was observed in cells expressing the caged T7RNAp, with a tunable linear response of gene expression to an increasing irradiation time ( Figure 3B).…”

“…A light-activated T7RNAp has previously been generated in our lab through the site-specific incorporation of an ortho-nitrobenzyl caged tyrosine (PCY) in E. coli cells with an expanded genetic code. 39 However, its application for the photochemical control of transcription in mammalian cells was hampered since the M. janaschii tRNA synthetase/tRNA pair is not orthogonal in mammalian cells and transfection of the caged protein was required. To overcome this limitation, site-specific incorporation of a genetically encoded photocaged lysine (PCK) into T7RNAp through an engineered, fully orthogonal pyrrolysine synthetase/tRNA pair was used to photochemically control RNA polymerization in mammalian cells with (Figure 1).…”

An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis

“…This contrasts to other genetic switches, such as the tetracycline-inducible system 47, 48 , that require the addition of a small molecule or hormone 49, 50 to activate gene expression. Photocaged unnatural amino acids or chemical inducers have also enabled a variety of approaches to inducible transcription and recombination controlled by UV light 12, 13, 51–54 . However, these methods currently require partial amber stop codon suppression to direct the incorporation of the photoreactive residue 55 .…”

“…Most of these systems use light-inducible, noncovalent protein-protein interactions that are reversible and allow for dynamic control of gene expression, such that gene activation or repression is always dependent on illumination 3–11 . Other systems use irreversible, covalent protein modifications, but the effect only lasts for the lifetime of the modified proteins 12, 13 . However, many applications require long-term changes in gene expression to achieve desired outcomes, and the necessity to maintain the exogenous illumination stimulus can be prohibitive.…”

Cellular Delivery and Photochemical Activation of Antisense Agents through a Nucleobase Caging Strategy

“…Previously, we and others, have introduced caging groups to the photochemical control of a variety of biological macromolecules, including antisense agents, 23-27 DNA decoys, 28 triplex-forming DNA, 29 siRNAs, 30-32 and proteins. 33, 34 However, all reported reagents need to be either transfected or injected into cells and organisms.…”