Photolabile Group Research Articles

Conditionally Activated Cross-Linked crRNAs for CRISPR/Cas12a Based Nucleic Acid Detection.

CRISPR/Cas systems, particularly CRISPR/Cas12a, have revolutionized nucleic acid detection due to their exceptional specificity and sensitivity. However, CRISPR/Cas12a's cleavage activity can interfere with amplification processes, such as reverse transcription (RT) and isothermal amplification (e.g., RPA), potentially compromising detection sensitivity and accuracy. While modified CRISPR/Cas12a systems employing caging and decaging strategies have been developed to address this, these approaches typically require extensive optimization of photolabile groups and complex assay configurations. Here, we present a universal, photochemically controlled strategy for CRISPR/Cas12a-based detection that overcomes these challenges. Our approach involves cross-linking a polymeric crRNA with a photoresponsive cross-linker, effectively inactivating it during amplification and enabling rapid activation through brief light exposure to cleave the cross-linker and release active crRNA. This method obviates the need for labor-intensive optimizations and modifications, making it highly versatile and suitable for rapid, on-site detection applications. Our strategy demonstrates enhanced versatility and applicability, particularly for the immediate detection of newly emerging or unexpected nucleic acid sequences, supporting applications in pathogen detection, genetic screening, and point-of-care diagnostics.

MicroRNA-Triggered Programmable DNA-Encoded Pre-PROTACs for Cell-Selective and Controlled Protein Degradation.

Proteolysis-targeting chimeras (PROTACs) have accelerated drug development; however, some challenges still exist owing to their lack of tumor selectivity and on-demand protein degradation. Here, we developed a miRNA-initiated assembled pre-PROTAC (miRiaTAC) platform that enables the on-demand activation and termination of target degradation in a cell type-specific manner. Using miRNA-21 as a model, we engineered DNA hairpins labeled with JQ-1 and pomalidomide and facilitated the modular assembly of DNA-encoded pre-PROTACs through a hybridization chain reaction. This configuration promoted the selective polyubiquitination and degradation of BRD4 upon miR-21 initiation, highlighting significant tumor selectivity and minimal systemic toxicity. Furthermore, the platform incorporates photolabile groups, enabling the precise optical control of pre-PROTACs during DNA assembly/disassembly, mitigating the risk of excessive protein degradation. Additionally, by introducing a secondary ligand targeting CDK6, these pre-PROTACs were used as a modular scaffold for the programmable assembly of active miRiaTACs containing two different warheads in exact stoichiometry, enabling orthogonal multitarget degradation. The integration of near-infrared light-mediated photodynamic therapy through an upconversion nanosystem further enhanced the efficacy of the platform with potent in vivo anticancer activity. We anticipate that miRiaTAC represents a significant intersection between dynamic DNA nanotechnology and PROTAC, potentially expanding the versatility of PROTAC toolkit for cancer therapy.

MicroRNA‐Triggered Programmable DNA‐Encoded Pre‐PROTACs for Cell‐Selective and Controlled Protein Degradation

AbstractProteolysis‐targeting chimeras (PROTACs) have accelerated drug development; however, some challenges still exist owing to their lack of tumor selectivity and on‐demand protein degradation. Here, we developed a miRNA‐initiated assembled pre‐PROTAC (miRiaTAC) platform that enables the on‐demand activation and termination of target degradation in a cell type‐specific manner. Using miRNA‐21 as a model, we engineered DNA hairpins labeled with JQ‐1 and pomalidomide and facilitated the modular assembly of DNA‐encoded pre‐PROTACs through a hybridization chain reaction. This configuration promoted the selective polyubiquitination and degradation of BRD4 upon miR‐21 initiation, highlighting significant tumor selectivity and minimal systemic toxicity. Furthermore, the platform incorporates photolabile groups, enabling the precise optical control of pre‐PROTACs during DNA assembly/disassembly, mitigating the risk of excessive protein degradation. Additionally, by introducing a secondary ligand targeting CDK6, these pre‐PROTACs were used as a modular scaffold for the programmable assembly of active miRiaTACs containing two different warheads in exact stoichiometry, enabling orthogonal multitarget degradation. The integration of near‐infrared light‐mediated photodynamic therapy through an upconversion nanosystem further enhanced the efficacy of the platform with potent in vivo anticancer activity. We anticipate that miRiaTAC represents a significant intersection between dynamic DNA nanotechnology and PROTAC, potentially expanding the versatility of PROTAC toolkit for cancer therapy.

Elucidating and Optimizing the Photochemical Mechanism of Coumarin-Caged Tertiary Amines.

Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φu < 1%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φu > 20%─a 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.

Evaluation of Antibacterial Functionalized Dihydropyrimidine Photoaffinity Probes Toward Mechanism of Action Studies.

Antibiotic-resistant bacteria are a global health concern, necessitating the development of antibiotics working through new or underutilized mechanisms. Functionalized amino dihydropyrimidines have previously demonstrated potential as antibacterial agents, but they had limited potency, and their biological mechanism was not understood. To further evaluate their potential, focused libraries were prepared and screened for bacterial growth inhibition, and these compounds provided additional insights into the structure-activity relationships, allowing for the preparation of compounds that inhibited all strains of Staphylococcus aureus with an MIC of 2 μg/mL. After eliminating the proposed mechanism of dihydrofolate reductase inhibition, trifluoromethyl diazirine photoaffinity probes were synthesized to investigate their mechanism, and these were tested to ensure the photolabile group did not impact the antibacterial activity. Finally, the compounds were screened for hemolysis and mammalian cytotoxicity. While they lacked nonspecific membrane rupturing activity, many of the compounds showed significant mammalian cytotoxicity, indicating further development will be required to render them selective for bacteria.

Phenylsulfonate as a photolabile group for intramolecular carbon-carbon cross-coupling reactions.

Efficient cyclization reactions play a pivotal role in the synthesis of extended polycyclic aromatic hydrocarbons (PAHs) and graphene nanoribbons. Although efficient reactions have been developed, a simple yet versatile method that is compatible with most functional groups is still lacking. Herein, we report the use of phenylsulfonates as a photolabile group to generate aryl radicals that undergo a radical cyclization reaction to produce triphenylene derivatives. The phenylsulfonate group proves to be a highly adaptable and robust photolabile group, and compatible with Suzuki cross-coupling conditions. Kinetic and optimization experiments have been conducted, shedding light on the potential of this reaction as a versatile tool for the synthesis of PAHs.

A photocaged, pH-sensitive anion transporter with AND logic dual-stimuli activation.

A pH-switchable anion transporter 1 was photocaged with two photolabile groups to enhance spatiotemporal control over its chloride transport activity. Simultaneous application of light irradiation and acidic pH restores the activity of 1, while either stimulus alone results in no or very low activity. The double activation strategy described herein has potential to yield more selectively cytotoxic anionophores for future medical applications.

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells.

Dynamic monitoring of intracellular ubiquitin (Ub) conjugates is instrumental to understanding the Ub regulatory machinery. Although many biochemical approaches have been developed to characterize protein ubiquitination, chemical tools capable of temporal resolution probing of ubiquitination events remain to be developed. Here, we report the development of the first cell-permeable and stimuli-responsive Ub probe and its application for the temporal resolution profiling of ubiquitinated substrates in live cells. The probe carrying the photolabile group N-(2-nitrobenzyl)-Gly (Nbg) on the amide bond between Ub Gly75 and Gly76 is readily prepared through chemical synthesis and can be delivered to live cells by conjugation via a disulfide bond with the cyclic cell-penetrating peptide cR10D (i.e., 4-((4-(dimethylamino)phenyl)-azo)-benzoic acid-modified cyclic deca-arginine). Both in vitro and in vivo experiments showed that Ub-modifying enzymes (E1, E2s, and E3s) could not install the Ub probe onto substrate proteins prior to removal of the nitrobenzyl group, which was easily accomplished via photoirradiation. The utility and practicality of this probe were exemplified by the time-resolved biochemical and proteomic investigation of ubiquitination events in live cells during a H2O2-mediated oxidative stress response. This work shows a conceptually new family of chemical Ub tools for the time-resolved studies on dynamic protein ubiquitination in different biological processes and highlights the utility of modern chemical protein synthesis in obtaining custom-designed tools for biological studies.

The Light-Controlled Release of 2-fluoro-l-fucose, an Inhibitor of the Root Cell Elongation, from a nitrobenzyl-caged Derivative.

Glycan metabolic engineering is a powerful tool for studying the glycosylation in living plant cells. The use of modified monosaccharides such as deoxy or fluorine-containing glycosides has been reported as a powerful pharmacological approach for studying the carbohydrate metabolism. 1,3,4-tri-O-acetyl-2-fluoro-l-fucose (2F-Fuc) is a potent inhibitor of the plant cell elongation. After feeding plant seedlings with 2F-Fuc, this monosaccharide derivative is deacetylated and converted by the endogenous metabolic machinery into the corresponding nucleotide-sugar, which then efficiently inhibits Golgi-localized fucosyltransferases. Among plant cell wall polymers, defects in the fucosylation of the pectic rhamnogalacturonan-II cause a decrease in RG-II dimerization, which in turn induce the arrest of the cell elongation. In order to perform the inhibition of the cell elongation process in a spatio-temporal manner, we synthesized a caged 3,4-di-O-acetyl-1-hydroxy-2-fluoro-l-fucose (1-OH-2F-Fuc) derivative carrying a photolabile ortho-nitrobenzyl alcohol function at the anomeric position: 3,4-di-O-acetyl-1-ortho-nitrobenzyl-2-fluoro-l-fucose (2F-Fuc-NB). The photorelease of the trapped 1-OH-2F-Fuc was performed under a 365 nm LED illumination. We demonstrated that the in planta elimination by photoexcitation of the photolabile group releases free 2F-Fuc in plant cells, which in turn inhibits in a dose-dependent manner and, reversibly, the root cell elongation.

Optically Responsive Protein Coating of DNA Origami for Triggered Antigen Targeting.

DNA nanostructures have emerged as modular building blocks in several research fields including biomedicine and nanofabrication. Their proneness to degradation in various environments has led to the development of a variety of nature-inspired protection strategies. Coating of DNA origami nanostructures with proteins can circumvent degradation and alter their properties. Here, we have used a single-chain variable antibody fragment and serum albumin to construct positively charged and stimuli-responsive protein-dendron conjugates, which were complexed with DNA origami through electrostatic interactions. Using a stepwise assembly approach, the coated nanostructures were studied for their interaction with the corresponding antigen in fluorescence-based immunoassays. The results suggest that the antibody–antigen interaction can be disturbed by the addition of the bulky serum albumin. However, this effect is fully reversible upon irradiation of the structures with an optical stimulus. This leads to a selective dissociation of the serum albumin from the nanostructure due to cleavage of a photolabile group integrated in the dendron structure, exposing the antibody fragment and enabling triggered binding to the antigen, demonstrating that serum albumin can be considered as an externally controlled “camouflaging” agent. The presented stimuli-responsive complexation approach is highly versatile regarding the choice of protein components and could, therefore, find use in DNA origami protection, targeting, and delivery as well as their spatiotemporal control.

Site-specific photolabile roadblocks for the study of transcription elongation in biologically complex systems

Transcriptional pausing is crucial for the timely expression of genetic information. Biochemical methods quantify the half-life of paused RNA polymerase (RNAP) by monitoring restarting complexes across time. However, this approach may produce apparent half-lives that are longer than true pause escape rates in biological contexts where multiple consecutive pause sites are present. We show here that the 6-nitropiperonyloxymethyl (NPOM) photolabile group provides an approach to monitor transcriptional pausing in biological systems containing multiple pause sites. We validate our approach using the well-studied his pause and show that an upstream RNA sequence modulates the pause half-life. NPOM was also used to study a transcriptional region within the Escherichia coli thiC riboswitch containing multiple consecutive pause sites. We find that an RNA hairpin structure located upstream to the region affects the half-life of the 5′ most proximal pause site—but not of the 3′ pause site—in contrast to results obtained using conventional approaches not preventing asynchronous transcription. Our results show that NPOM is a powerful tool to study transcription elongation dynamics within biologically complex systems.

Ligand-Directed Modification of Active Matrix Metalloproteases: Activity-based Probes with no Photolabile Group.

Activity-based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non-covalent, activity-based probes targeting them have been systematically developed by decorating reversible inhibitors with photo-crosslinkers. By exploiting two types of ligand-guided chemistry, we identified novel activity-based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S3 ' region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand-directed chemistry was successfully applied to label active MMP-12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo.

Ligand‐Directed Modification of Active Matrix Metalloproteases: Activity‐based Probes with no Photolabile Group

AbstractActivity‐based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non‐covalent, activity‐based probes targeting them have been systematically developed by decorating reversible inhibitors with photo‐crosslinkers. By exploiting two types of ligand‐guided chemistry, we identified novel activity‐based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S3′ region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand‐directed chemistry was successfully applied to label active MMP‐12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo.

Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.

Break Up to Make Up: Utilization of Photocleavable Groups in Biolabeling of Hydrogel Scaffolds

AbstractUnderstanding cell behavior within the extracellular matrix is essential for the implementation of biomaterials in medical applications. Conjugation of bioactive molecules to hydrogel scaffolds imparts the materials with biological cues necessary for controlling cell behavior such as adhesion, growth and migration. Photo‐induced ligation is a powerful tool to precisely control when and where such bioactivity can be initiated. Over the past decade, advances in light‐regulated technology have emerged to address the stringent demands for photochemical ligation in biological environments, including high sensitivity, fast reaction kinetics, longer wavelength absorption and site‐selectivity. Here, the utilization of contemporary photolabile groups for light‐mediated bioconjugation of hydrogels are highlighted. In this approach, reactive groups such as thiol, amine and aldehyde are masked with photolabile moieties, which can be photo‐released to participate in click reactions, such as Michael addition and oxime ligation. New chromophores with long wavelength visible light absorption are also examined for potential employment in light‐induced bioconjugation.

Substrate-Photocaged Enzymatic Fluorogenic Probe Enabling Sequential Activation for Light-Controllable Monitoring of Intracellular Tyrosinase Activity.

Tyrosinase (TYR) is a crucial enzyme involved in melanogenesis, and its overexpression is closely associated with melanoma. To precisely monitor intracellular TYR activity, remote control of a molecule imaging tool is highly meaningful but remains to be explored. In this work, we present the first photocaged tyrosinase fluorogenic probe by caging the substrate of the enzymatic probe with a photolabile group. Because of the sequential light and enzyme-activation feature, this probe exhibits photocontrollable "turn on" response toward TYR with good selectivity and high sensitivity (detection limit: 0.08 U/mL). Fluorescence imaging results validate that the caged probe possesses the capability of visualizing intracellular endogenous tyrosinase activity in a photocontrol fashion, thus offering a promising molecule imaging tool for investigating TYR-related physiological function and pathological role. Moreover, our sequential activation strategy has great potential for developing more photocontrollable enzymatic fluorogenic probes with spatiotemporal resolution.

Sanger's Reagent Sensitized Photocleavage of Amide Bond for Constructing Photocages and Regulation of Biological Functions.

Photolabile groups offer promising tools to study biological processes with high spatial and temporal control. In the investigation, we designed and prepared several new glycine amide derivatives of Sanger's reagent and demonstrated that they serve as a new class of photocages for Zn2+ and an acetylcholinesterase (AChE) inhibitor. We showed that the mechanism for photocleavage of these substances involves initial light-driven cyclization between the 2,4-dinitrophenyl and glycine methylene groups to form acyl benzimidazole N-oxides, which undergo secondary photoinduced decarboxylation in association with rupture of an amide bond. The cleavage reactions proceed with modest to high quantum yields. We demonstrated that these derivatives can be used in targeted intracellular delivery of Zn2+, fluorescent imaging by light-triggered Zn2+ release, and regulation of biological processes including the enzymatic activity of carbonic anhydrase (CA), negative regulation of N-methyl-d-aspartate receptors (NMDARs), and pulse rate of cardiomyocytes. The successful proof-of-concept examples described above open a new avenue for using Sanger's reagent-based glycine amides as photocages for the exploration of complex cellular functions and signaling pathways.

Synthesis of photolabile group protected anomeric acetals and its application in carbohydrate synthesis with the assistance of continuous flow photo-reactor

Synthesis of photolabile group protected anomeric acetals and its application in carbohydrate synthesis with the assistance of continuous flow photo-reactor

Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA.

The function of CRISPR/Cas9 can be conditionally controlled by the rational engineering of guide RNA (gRNA) to target the gene of choice for precise manipulation of the genome. Particularly, chemically modified gRNA that can be activated by using specific stimuli provides a unique tool to expand the versatility of conditional control. Herein, unlike previous engineering of gRNA that generally focused on the RNA part only but neglected RNA–protein interactions, we aimed at the interactive sites between 2′-OH of ribose in the seed region of gRNA and the Cas9 protein and identified that chemical modifications at specific sites could be utilized to regulate the Cas9 activity. By introducing a photolabile group at these specific sites, we achieved optical control of Cas9 activity without disrupting the Watson–Crick base pairing. We further examined our design through CRISPR-mediated gene activation and nuclease cleavage in living cells and successfully manipulated the gene expression by using light irradiation. Our site-specific modification strategy exhibited a highly efficient and dynamic optical response and presented a new perspective for manipulating gRNA based on the RNA–protein interaction rather than the structure of RNA itself. In addition, these specific sites could also be potentially utilized for modification of other stimuli-responsive groups, which would further enrich the toolbox for conditional control of CRISPR/Cas9 function.

Chemoselective Derivatization of Folded Synthetic Insulin Variants with Potassium Acyltrifluoroborates (KATs)

AbstractSynthetic folded insulin variants containing an ornithine‐hydroxylamine residue are readily modified in aqueous buffers by amide‐forming ligations with potassium acyltrifluoroborates (KATs). The synthetic insulin analogs were prepared by Fmoc‐SPPS, α‐ketoacid‐hydroxylamine (KAHA) ligation, and a prosthetic C‐peptide that delivers the correct disulfide pattern and allows facile incorporation at the B0 position of Glargine M2 of a new ornithine hydroxylamine protected with a photolabile group. The folded insulin is readily modified by photo‐deprotection followed by amide‐forming KAT ligation to give insulin variants labeled with dyes, lipids, and PEGs, as well as the formation of a covalent dimer.