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

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.     

Optical Control of Cellular ATP Levels with a Photocaged Adenylate Kinase

We have developed a new tool for the optical control of cellular ATP concentrations with a photocaged adenylate kinase (Adk). The photocaged Adk is generated by substituting a catalytically essential lysine with a hydroxycoumarin-protected lysine through site-specific unnatural amino acid mutagenesis in both E. coli and mammalian cells. Caging of the critical lysine residue offers complete suppression of Adk's phosphotransferase activity and rapid restoration of its function both in vitro and in vivo upon optical stimulation. Light-activated Adk renders faster rescue of cell growth than chemically inducible expression of wild-type Adk in E. coli as well as rapid ATP depletion in mammalian cells. Thus, caging Adk provides a new tool for direct conditional perturbation of cellular ATP concentrations thereby enabling the investigation of ATP-coupled physiological events in temporally dynamic contexts.     

Optochemical Control of Bacterial Gene Expression: Novel Photocaged Compounds for Different Promoter Systems

Photocaged compounds are applied for implementing precise, optochemical control of gene expression in bacteria. To broaden the scope of UV-light-responsive inducer molecules, six photocaged carbohydrates were synthesized and photochemically characterized, with the absorption exhibiting a red-shift. Their differing linkage through ether, carbonate, and carbamate bonds revealed that carbonate and carbamate bonds are convenient. Subsequently, those compounds were successfully applied in vivo for controlling gene expression in E. coli via blue light illumination. Furthermore, benzoate-based expression systems were subjected to light control by establishing a novel photocaged salicylic acid derivative. Besides its synthesis and in vitro characterization, we demonstrate the challenging choice of a suitable promoter system for light-controlled gene expression in E. coli. We illustrate various bottlenecks during both photocaged inducer synthesis and in vivo application and possibilities to overcome them. These findings pave the way towards novel caged inducer-dependent systems for wavelength-selective gene expression.     

Controlling PROTACs with Light

Proteolysis targeting chimeras, PROTACs, are emerging as a powerful strategy for exerting exogenous control over protein levels, allowing small molecules to exploit the ubiquitin–proteasome pathway for targeted protein degradation. This highlight focuses on the fusion of photochemistry with these bifunctional compounds, which has provided a novel pathway for spatiotemporally tuning the activation of PROTACs in the form of their photocaged and photoswitchable versions. Photocaged PROTACs consist of a hindered optolabile group that detaches only upon irradiation at a specific wavelength, releasing the active PROTAC. These modified PROTACs are inactive in the dark. Photoswitchable PROTACs are photoisomerizable molecules with azobenzene linkages that are active in either the cis or trans form and inactive in the other. The isomers interconvert upon irradiation with an appropriate wavelength of light and relax to the thermodynamically stable isomer in the dark or with another wavelength of light. Although photocaged PROTACs only permit activation control for protein degradation, photoswitching PROTACs offer reversible activation and deactivation by using suitable wavelengths of light.     

Genetic Encoding of Photocaged Tyrosines with Improved Light‐Activation Properties for the Optical Control of Protease Function

We genetically encoded three new caged tyrosine analogues with improved photochemical properties by using an engineered pyrrolysyl‐tRNA synthetase/tRNA_CUA pair in bacterial and mammalian cells. We applied the new tyrosine analogues to the photoregulation of firefly luciferase by caging its key tyrosine residue, Tyr340, and observed excellent off‐to‐on light switching. This reporter was then used to evaluate the activation rates of the different light‐removable protecting groups in live cells. We identified the nitropiperonyl caging group as an excellent compromise between incorporation efficiency and photoactivation properties. To demonstrate applicability of the new caged tyrosines, an important proteolytic enzyme, tobacco etch virus (TEV) protease, was engineered for optical control. The ability to incorporate differently caged tyrosine analogues into proteins in live cells further expands the unnatural amino acid and optogenetic toolbox.     

Photocaged Hoechst Enables Subnuclear Visualization and Cell Selective Staining of DNA in vivo

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.     

Ergothioneine Biosynthetic Methyltransferase EgtD Reveals the Structural Basis of Aromatic Amino Acid Betaine Biosynthesis

Ergothioneine is an N‐α‐trimethyl‐2‐thiohistidine derivative that occurs in human, plant, fungal, and bacterial cells. Biosynthesis of this redox‐active betaine starts with trimethylation of the α‐amino group of histidine. The three consecutive methyl transfers are catalyzed by the S‐adenosylmethionine‐dependent methyltransferase EgtD. Three crystal structures of this enzyme in the absence and in the presence of N‐α‐dimethylhistidine and S‐adenosylhomocysteine implicate a preorganized array of hydrophilic interactions as the determinants for substrate specificity and apparent processivity. We identified two active site mutations that change the substrate specificity of EgtD 10⁷‐fold and transform the histidine‐methyltransferase into a proficient tryptophan‐methyltransferase. Finally, a genomic search for EgtD homologues in fungal genomes revealed tyrosine and tryptophan trimethylation activity as a frequent trait in ascomycetous and basidomycetous fungi.     

A Light-Activatable Photocaged Variant of the Ultra-High Affinity ALFA-Tag Nanobody

Nanobodies against short linear peptide-epitopes are widely used to detect and bind proteins of interest (POI) in fusion constructs. Engineered nanobodies that can be controlled by light have found very recent attention for various extra- and intracellular applications. We here report the design of a photocaged variant of the ultra-high affinity ALFA-tag nanobody, also termed ALFA-tag photobody. ortho-Nitrobenzyl tyrosine was incorporated into the paratope region of the nanobody by genetic code expansion technology and resulted in a ≥9,200 to 100,000-fold impairment of the binding affinity. Irradiation with light (365 nm) leads to decaging and reconstitutes the native nanobody. We show the light-dependent binding of the ALFA-tag photobody to HeLa cells presenting the ALFA-tag. The generation of the first photobody directed against a short peptide epitope underlines the generality of our photobody design concept. We envision that this photobody will be useful for the spatiotemporal control of proteins in many applications using cultured cells.     

Hacking the Genetic Code of Mammalian Cells

A genetic shuttle : The highlighted article, which was recently published by Schultz, Geierstanger and co‐workers, describes a straightforward scheme for enlarging the genetic code of mammalian cells. An orthogonal tRNA/aminoacyl‐tRNA synthetase pair specific for a new amino acid can be evolved in E. coli and subsequently transferred into mammalian cells. The feasibility of this approach was demonstrated by adding a photocaged lysine derivative to the genetic repertoire of a human cell line.

     

Spatio-Temporal Photoactivation of Cytotoxic Proteins

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.     

Optical Control of DNA Helicase Function through Genetic Code Expansion

Nucleotide excision repair (NER) is a general DNA repair mechanism that is capable of removing a wide variety of DNA lesions induced by physical or chemical insults. UvrD, a member of the helicase SF1 superfamily, plays an essential role in bacterial NER by unwinding the duplex DNA in the 3′ to 5′ direction to displace the lesion‐containing strand. In order to achieve conditional control over NER, we generated a light‐activated DNA helicase. This was achieved through a site‐specific incorporation of a genetically encoded hydroxycoumarin lysine at a crucial position in the ATP‐binding pocket of UvrD. The resulting caged enzyme was completely inactive in several functional assays. Moreover, enzymatic activity of the optically triggered UvrD was comparable to that of the wild‐type protein, thus demonstrating excellent OFF to ON switching of the helicase. The developed approach provides optical control of NER, thereby laying a foundation for the regulation of ATP‐dependent helicase functions in higher organisms. In addition, this methodology is applicable to the light‐activation of a wide range of ATPases.     

Specific Modulation of Protein Activity by Using a Bioorthogonal Reaction

Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a “click” reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z‐containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active‐site occlusion and allosteric mechanisms. We have termed this a “clickable magic bullet” strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.     

Interrogating PP1 Activity in the MAPK Pathway with Optimized PP1-Disrupting Peptides

Protein phosphatase-1 (PP1)-disrupting peptides (PDPs) are selective chemical modulators of PP1 that liberate the active PP1 catalytic subunit from regulatory proteins; thus allowing the dephosphorylation of nearby substrates. We have optimized the original cell-active PDP3 for enhanced stability, and obtained insights into the chemical requirements for stabilizing this 23-mer peptide for cellular applications. The optimized PDP-Nal was used to dissect the involvement of PP1 in the MAPK signaling cascade. Specifically, we have demonstrated that, in human osteosarcoma (U2OS) cells, phosphoMEK1/2 is a direct substrate of PP1, whereas dephosphorylation of phosphoERK1/2 is indirect and likely mediated through enhanced tyrosine phosphatase activity after PDP-mediated PP1 activation. Thus, as liberators of PP1 activity, PDPs represent a valuable tool for identifying the substrates of PP1 and understanding its role in diverse signaling cascades.     

Photoactivatable Mussel‐Based Underwater Adhesive Proteins by an Expanded Genetic Code

Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4‐dihydroxyphenylalanine (DOPA)‐rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl‐transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho‐nitrobenzyl DOPA (ONB‐DOPA). The engineered ONB‐DOPARS enables in vivo production of MAP type 5 site‐specifically equipped with multiple instances of ONB‐DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives.     

Efficient Incorporation of Clickable Unnatural Amino Acids Enables Rapid and Biocompatible Labeling of Proteins in Vitro and in Bacteria

Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment – typically at a single site or at a few sites per protein – of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l -phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production – using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain – of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling.     

The Red‐/Green‐Switching GAF3 of Cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803 Regulates the Activity of an Adenylyl Cyclase

Cyanobacteriochromes (CBCRs) are photoreceptors in cyanobacteria that present a bilin chromophore‐binding GAF domain as a photochromic element to control the activity of a downstream enzyme or regulator. CBCR Slr1393 from Synechocystis PCC 6803 carries three GAF domains, but only the third one binds phycocyanobilin covalently. Slr1393 shows photochromicity between red and green absorbing states and regulates a C‐terminally located histidine kinase. In this work, we fused this third GAF domain to an adenylyl cyclase (AC) from Microcoleus chthonoplastes PCC7420 that in its genuine form is under blue‐light control from a LOV domain. A series of RGS‐AC variants were constructed with various lengths of the linkers between RGS and AC. Assays in vitro and in living Escherichia coli cells (AC‐deletion mutant) demonstrated that the activity of AC was light regulated, namely, the red‐light‐converted form of RGSΔ14‐Δ4AC (in vitro) was about three times more active than the green‐light‐converted form. Expression of the fusion protein RGSΔ14‐Δ4AC in vivo again showed highest light regulation with at least threefold amplification of the AC function. In some experiments, even tenfold higher activity was observed, which indicated that the protein, if expressed under in vivo conditions, was part of the E. coli physiological conditions and thereby subjected to more complex and variable regulation through other E. coli inherent factors.     

Light-Wavelength-Based Quantitative Control of Dihydrofolate Reductase Activity by Using a Photochromic Isostere of an Inhibitor

Photopharmacology has attracted research attention as a new tool for achieving optical control of biomolecules, following the methods of caged compounds and optogenetics. We have developed an efficient photopharmacological inhibitor—azoMTX—for Escherichia coli dihydrofolate reductase (eDHFR) by replacing some atoms of the original ligand, methotrexate, to achieve photoisomerization properties. This fine molecular design enabled quick structural conversion between the active “bent” Z isomer of azoMTX and the inactive “extended” E isomer, and this property afforded quantitative control over the enzyme activity, depending on the wavelength of irradiating light applied. Real-time photoreversible control over enzyme activity was also achieved.     

Plasmid DNA-Based Bioluminescence-Activated System for Photodynamic Therapy in Cancer Treatment

The low depth of tissue penetration by therapeutic light sources severely restricts photodynamic therapy (PDT) in treating deep-seated tumors. Using a luciferase/d -luciferin bioluminescence system to artificially create internal light sources in cells instead of external light sources is an effective means of solving the above problems. However, high-efficiency bioluminescence requires a higher concentration of luciferase in the cell, which poses a considerable challenge to the existing system of enzyme loading, delivery, activity and retention of drugs, and dramatically increases the cost of treatment. We loaded the substrate D-luciferin, and the photosensitizer hypericin into a polyethyleneimine (PEI)-modified nano-calcium phosphate (CaP) to solve this problem. Subsequently, the plasmid DNA containing the luciferase gene was loaded onto it using the high-density positive charge characteristic of PEI from the nanodrug (denoted DHDC). After the DHDC enters the tumor cell, it collapses and releases the plasmid DNA, which uses the intracellular protein synthesis system to continuously and massively express luciferase. Using endogenous ATP, Mg²⁺, and O₂ in cells, luciferase oxidizes d -luciferin and produces luminescence. The luminescence triggers hypericin excitation to generate ROS and kill cancer cells. This study provides a new strategy for the application of bioluminescence in PDT treatment.