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Identification of Protein Targets of Bioactive Small Molecules Using Randomly Photomodified Probes
摘要： Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe re-synthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.
关键词： bioactive small molecules,target isolation,protein targets,target identification,stochastic modification,target visualization,photoactivatable phenyldiazirine linker,biotin,fluorophore,hydrophilic copolymer
更新于2025-09-23 15:21:21
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Microscope laser assisted photooxidative activation of bioorthogonal ClickOx probes
摘要： A photoactivatable fluorogenic tetrazine-rhodaphenothiazine probe was synthesized and studied in light-assisted, bioorthogonal labeling schemes. Experimental results revealed that the bioorthogonally conjugated probe efficiently sensitizes 1O2 generation upon illumination with green or orange light and undergoes self-oxidation leading to an intensely fluorescent sulfoxide product. An added value of the present probe is that it is also suitable for STED super-resolution microscopy using a 660 nm depletion laser.
关键词： tetrazine-rhodaphenothiazine,fluorogenic,STED,super-resolution microscopy,photoactivatable,bioorthogonal,labeling
更新于2025-09-23 15:19:57
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Photoactivatable Prodrug-Backboned Polymeric Nanoparticles for Efficient Light-Controlled Gene Delivery and Synergistic Treatment of Platinum-Resistant Ovarian Cancer
摘要： Combination of chemotherapy and gene therapy provides an effective strategy for cancer treatment. However, lacking of suitable co-delivery systems with efficient endo/lysosomal escape and controllable drug release/gene unpacking is the major bottleneck for maximizing the combinational therapeutic efficacy. Herein, we developed a photoactivatable Pt(IV) prodrug-backboned polymeric nanoparticles system (CNPPtCP/si(c-fos)) for light-controlled si(c-fos) delivery and synergistic photoactivated chemotherapy (PACT) and RNAi on platinum-resistant ovarian cancer (PROC). Upon blue light irradiation (430 nm), CNPPtCP/si(c-fos) could generate oxygen-independent N3? with mild oxidation energy for efficient endo/lysosomal escape through N3?-assisted photochemical internalization with less gene deactivation. Thereafter, along with Pt(IV) prodrug activation, CNPPtCP/si(c-fos) would be disassociated to release active Pt(II) and unpack si(c-fos) simultaneously. Both in vitro and in vivo results demonstrated that CNPPtCP/si(c-fos) displayed excellent synergistic therapeutic efficacy on PROC with low toxicity. This PACT prodrug-backboned polymeric nanoplatform may provide a promising gene/drug co-delivery tactics for treatment of various hard-to-tackle cancers.
关键词： N3?-assisted photochemical internalization,photoactivatable polymeric nanoparticles,gene delivery,platinum-resistant ovarian cancer,photoactivated chemotherapy
更新于2025-09-19 17:13:59
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Green-Light-Induced PhotoCORM: Lysozyme Binding Affinity towards Mn <sup>I</sup> and Re <sup>I</sup> Carbonyl Complexes and Biological Activity Evaluation
摘要： Reaction of N-(2-pyridylmethylene)benzene-1,4-diamine (L) with [MBr(CO)5] (M = MnI and ReI) affords complexes of the type [MBr(CO)3L] [M = MnI (1) and ReI (2)]. Complex 1 releases CO upon illumination with light in the range of 525–468 nm. No photo induced CO release is detected from 2. Reactive azide complex 3, obtained by bromide's ligand exchange, reacts with the electron-poor alkyne dimethyl acetylene dicarboxylate giving triazolato complex 4 ([Mn(triazolateCOOMe,COOMe)(CO)3L]) via the free catalyst [3+2] cycloaddition coupling. Complex 1 exhibits interesting antifungal activity (MIC = 30 nM) against Candida albicans and Cryptococcus neoformans as well as cytotoxic activity of 12.56 μg/mL against noncancerous human embryonic kidney cells. Reactivity of the complexes towards hen egg white lysozyme is studied by electrospray ionization mass spectrometry.
关键词： Carbon monoxide,Biological,Photoactivatable,TDDFT,Lysozyme,binding affinity
更新于2025-09-10 09:29:36
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Photochemically Active Dyes for Super-Resolution Microscopy
摘要： The development of super-resolved optical microscopies has revolutionized the way we visualize cell biology. These techniques strongly rely on the use of photochemically active fluorophores that display changes in their photophysical properties upon irradiation with light. Many reversible and irreversible photochemical transformations have been explored for this purpose, and different imaging techniques require specific mechanisms of photoconversion. In this review, we provide an overview of the most common strategies used for the development of fluorophores for super-resolution microscopy and give specific examples of state-of-the-art fluorogenic probes. Furthermore, we discuss their main field of application and possible directions for future developments.
关键词： Live-cell imaging,Photoswitchable,Super-resolution microscopy,Fluorescent probes,Photoactivatable
更新于2025-09-09 09:28:46
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Choosing the right label for single-molecule tracking in live bacteria: side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes
摘要： Visualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins (PA-FPs). An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and results from single-molecule localization and tracking experiments. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.
关键词： Halo tag,single-molecule tracking,photoactivatable fluorescent protein,Escherichia coli,fluorophores,super-resolution microscopy,DNA-binding proteins
更新于2025-09-04 15:30:14