Research progress of hydrogen sulfide fluorescent probes targeting organelles.

Hydrogen sulfide (H2S) is implicated in numerous physiological and pathological processes in living organisms. Abnormal levels of H2S can result in various physiological disorders, highlighting the crucial need for effective identification and detection of H2S at the organellar level. Although numerous H2S fluorescent probes targeting organelles have been reported, a comprehensive review of these probes is required. This review focuses on the strategic selection of organelle-targeting groups and recognition sites for H2S fluorescent probes. This review examines H2S fluorescent probes that can specifically target lysosomes, mitochondria, endoplasmic reticulum, Golgi apparatus, and lipid droplets. These fluorescent probes have been meticulously classified and summarized based on their distinct targets, emphasizing their chemical structure, reaction mechanisms, and biological applications. We carefully designed fluorescent probes to efficiently enhance their ability to recognize target substances and exhibit significant fluorescence variations. Furthermore, we discuss the challenges inherent in the development of fluorescent probes and outline potential future directions for this exciting field.

Near-infrared fluorescent probe visual detection of Hg2+ and its application in biological system and ecological system.

Mercury ion (Hg2+), a heavy metal cation with greater toxicity, is widely present in the ecological environment and has become a serious threat to human health and environmental safety. Currently, developing a solution to simultaneously visualize and monitor Hg2+ in environmental samples, including water, soil, and plants, remains a great challenge. In this work, we created and synthesized a near-infrared fluorescent probe, BBN-Hg, and utilized Hg2+ to trigger the partial cleavage of the carbon sulfate ester in BBN-Hg as a sensing mechanism, and the fluorescence intensity of BBN-Hg was significantly enhanced at 650 nm, thus realizing the visualization of Hg2+ with good selectivity (detection limit, 53 nM). In live cells and zebrafish, the probe BBN-Hg enhances the red fluorescence signal in the presence of Hg2+, and successfully performs 3D imaging on zebrafish, making it a powerful tool for detecting Hg2+ in living systems. More importantly, with BBN-Hg, we are able to detect Hg2+ in actual water samples, soil and plant seedling roots. Furthermore, the probe was prepared as a test strip for on-site determination of Hg2+ with the assistance of a smartphone. Therefore, this study offers an easy-to-use and useful method for tracking Hg2+ levels in living organisms and their surroundings.

Hemiporphycene: A pH-tunable specific probe for Zn2+ and Cu2.

Porphyrins and their isomers possess high affinity towards the formation of complexes with metal cations, but their use for the determination of metal cations is rather limited, due to low selectivity. In this study, we have investigated the unsubstituted hemiporphycene, which shows a highly irregular shape of the inner cavity, and very different reactivity with various metal cations in methanol:water solutions. It was found that hemiporphycene can act as a pH-tunable specific probe for the determination of Zn2+ at pH 8.6 and specific for Cu2+ detection at pH 5.5.

Click Chemistry in Detecting Protein Modification.

Click chemistry, also known as "link chemistry," is an important molecular connection method that can achieve simple and efficient connections between specific small molecular groups at the molecular level. Click chemistry offers several advantages, including high efficiency, good selectivity, mild conditions, and few side reactions. These features make it a valuable tool for in-depth analysis of various protein posttranslational modifications (PTMs) caused by changes in cell metabolism during viral infection. This chapter considers the palmitoylation, carbonylation, and alkylation of STING and presents detailed information and experimental procedures for measuring PTMs using click chemistry.

Small Molecule Quinoxaline Fluorescent Probe for AcO- Real-Time Detection in Vivo.

Fluorescent probes to detect biologically important acetate ion (AcO-) are essential for regulating substance metabolism, alleviating inflammatory symptoms, reducing cancer incidence, and diagnosing early diseases. However, the relatively small charge-to-atomic radius ratio in AcO- and its triangular spatial structure pose challenges in recognition and often lead to interference from other anions in detection methods. Herein, we introduce a quinoxaline fluorescent probe, o-(4-(2-(3-oxo-3,4 -dihydroqui-noxalin-2-yl)vinyl)phenyl) dimethylaminothiophene ester (QPDMT), specifically design and synthetic for the accurate detection of AcO-. This probe leverages molecular nucleophilicity and electron transfer to undergo a reaction that releases the fluorophore upon cleavage of the thioformyl ether bond, exhibiting a turn-on fluorescence response at 530 nm. QPDMT exhibits an impressively low detection limit of 30 nM, a rapid response time of 20 min, a robust linear response in the 1-9 µM range and excellent fluorescence quantum yield, 0.32. Importantly, this probe demonstrates low cytotoxicity, making it an ideal candidate for endogenous AcO- detection in living cells and organisms.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Fluorescence Polarization Assay for Infection Diagnostics: A Review.

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents.

Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates.

Here, we present a lysate-based split-luciferase assay for examining protein-protein interactions (PPIs) in HEK293T cell lysates, exemplified by interactions between subunits of protein phosphatase PP1. We describe steps for storing and re-using lysates, sensor design, assay setup/optimization, and high-throughput screening of compound libraries. We then detail procedures for applying the assay as a research tool to characterize the dynamics of PPIs, which we illustrate with specific examples. For complete details on the use and execution of this protocol, please refer to Claes and Bollen.1.

N-terminomics profiling of naïve and inflamed murine colon reveals proteolytic signatures of legumain.

Legumain is a cysteine protease broadly associated with inflammation. It has been reported to cleave and activate protease-activated receptor 2 to provoke pain associated with oral cancer. Outside of gastric and colon cancer, little has been reported on the roles of legumain within the gastrointestinal tract. Using a legumain-selective activity-based probe, LE28, we report that legumain is activated within colonocytes and macrophages of the murine colon, and that it is upregulated in models of acute experimental colitis. We demonstrated that loss of legumain activity in colonocytes, either through pharmacological inhibition or gene deletion, had no impact on epithelial permeability in vitro. Moreover, legumain inhibition or deletion had no obvious impacts on symptoms or histological features associated with dextran sulfate sodium-induced colitis, suggesting its proteolytic activity is dispensable for colitis initiation. To gain insight into potential functions of legumain within the colon, we performed field asymmetric waveform ion mobility spectrometry-facilitated quantitative proteomics and N-terminomics analyses on naïve and inflamed colon tissue from wild-type and legumain-deficient mice. We identified 16 altered cleavage sites with an asparaginyl endopeptidase signature that may be direct substrates of legumain and a further 16 cleavage sites that may be indirectly mediated by legumain. We also analyzed changes in protein abundance and proteolytic events broadly associated with colitis in the gut, which permitted comparison to recent analyses on mucosal biopsies from patients with inflammatory bowel disease. Collectively, these results shed light on potential functions of legumain and highlight its potential roles in the transition from inflammation to colorectal cancer.

Green synthesis of thiazole bis-imines as fluorometric sensor for determination of lead in environmental, biological, and food samples.

In this work, we describe for the first time the synthesis of a thiazole bis-imine fluorometric sensor for the selective determination of Pb2+ in environmental, biological, and food samples. The novel molecules were obtained through a multicomponent reaction using a green and environmentally sustainable methodology. Synthesized chemical sensors were characterized using spectroscopic techniques to structural elucidation, including UV-Vis, FTIR-ATR, 1H and 13C NMR. One of these sensors exhibited remarkable selectivity for the Pb2+ ion at pH 3, forming a stable 1:1 (metal:ligand) complex. Additionally, the reaction conditions for complex formation were optimized, resulting in a method with a linear range of 0.667-10  µg L-1 and a detection limit of 0.18  µg L-1. Furthermore, method validation reinforced its reliability, showing low relative standard deviation in both intra-day and inter-day analyses. Recovery experiments ranged from 83.53 % to 119.10 %. This study represents a significant and innovative advancement in the development of rapid, sensitive, and alternative methods for the detection of potentially toxic metals in a wide range of samples employing a green multicomponent reaction of thiazole bis-imines.

Thermodynamics and Kinetics-Directed Regulation of Nucleic Acid-Based Molecular Recognition.

Nucleic acid-based molecular recognition plays crucial roles in various fields like biosensing and disease diagnostics. To achieve optimal detection and analysis, it is essential to regulate the response performance of nucleic acid probes or switches to match specific application requirements by regulating thermodynamics and kinetics properties. However, the impacts of thermodynamics and kinetics theories on recognition performance are sometimes obscure and the relative conclusions are not intuitive. To promote the thorough understanding and rational utilization of thermodynamics and kinetics theories, this review focuses on the landmarks and recent advances of nucleic acid thermodynamics and kinetics and summarizes the nucleic acid thermodynamics and kinetics-based strategies for regulation of nucleic acid-based molecular recognition. This work hopes such a review can provide reference and guidance for the development and optimization of nucleic acid probes and switches in the future, as well as for advancements in other nucleic acid-related fields.

Mechanosensitive fluorescence lifetime probes for investigating the dynamic mechanism of ferroptosis.

Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.

A Carbazole-based Fluorescent Probe with AIE Performance and a Large Stokes Shift for Peroxynitrite Detection and Imaging in Live Cells.

A carbazole-based fluorescent probe YCN with AIE performance and a large Stokes shift (242 nm) shift was synthesized by attaching 4-acetonitrile pyridine to the 3-phenylaldehyde butylcarbazole. Its structure was characterized by 1H NMR, 13C NMR and MS. Probe YCN has high selectivity and sensitivity toward ONOO-. The addition of ONOO- to the probe YCN solution results in a noticeable color change from pale yellow to colorless under natural light, and a fluorescent color change from bright orange-yellow to bright yellow-green under a 365 nm UV lamp, which can be distinguished by the naked eye. The research results on the reaction mechanism showed that when YCN reacted with ONOO-, -C = C- was oxidized and broken into -CHO, and the ICT effect was significantly inhibited, resulting in changes in UV absorption and fluorescence emission phenomenon. The recognition mechanism was verified by 1H NMR, mass spectrometry (MS) and density function theory (DFT) calculations. The experiments of live cells imaging suggested that compound YCN can be used as a fluorescent probe for the detection of ONOO- in HeLa cells. This result indicates that YCN has potential application prospects in the biological aspects.

Covalent labeling of Matrix Metalloproteases with affinity-based probes containing tuned reactive N-acyl-N-alkyl sulfonamide cleavable linkers.

Original covalent probes with an N-acyl-N-alkyl sulfonamide cleavable linker were developed to target a broad set of human Matrix Metalloproteases (MMPs). The electrophilicity of this cleavable linker was modulated to improve the selectivity of the probes as well as reduce their unspecific reactivity in complex biological matrices. We first demonstrated that targeting the S3 subsite of MMPs enables access to broad-spectrum affinity-based probes that exclusively react with the active version of these proteases. The probes were further assessed in proteomes of varying complexity, where human MMP-13 was artificially introduced at known concentration and the resulting labeled MMP was imaged by in-gel fluorescence imaging. We showed that the less reactive probe was still able to covalently modify MMP-13 while exhibiting reduced off-target unspecific reactivity. This study clearly demonstrated the importance of finely controlling the reactivity of the NASA warhead to improve the selectivity of covalent probes in complex biological systems.

Establishing of 3D-FISH on frozen section and its applying in chromosome territories analysis in Populus trichocarpa.

KEY MESSAGE: Fluorescence in situ hybridization with frozen sections of root tips showed difference of chromosome territories distribution between autosome and sex-chromosome homologous pairs in Populus trichocarpa. The spatial organization of chromatin within the interphase nucleus and the interactions between chromosome territories (CTs) are essential for various biologic processes. Three-dimensional fluorescence in situ hybridization (3D-FISH) is a powerful tool for analyzing CTs, but its application in plants is limited. In this study, we established a 3D-FISH technique using frozen sections of Populus trichocarpa root tips, which was an improvement over the use of paraffin sections and enabled us to acquire good FISH signals. Using chromosome-specific oligo probes, we were able to analyze CTs in interphase nuclei in three dimensions. The distribution of chromosome pairs 17 and 19 in the 3D-preserved nuclei of P. trichocarpa root tip cells were analyzed and showed that the autosome pair 17 associated more often than sex chromosome 19. This research lays a foundation for further study of the spatial position of chromosomes in the nucleus and the relationship between gene expression and spatial localization of chromosomes in poplar.

Whole-Tumor Clearing and Imaging of Intratumor Microbiota in Three Dimensions with miCDaL Strategy.

Acquiring detailed spatial information about intratumor microbiota in situ is challenging, which leaves 3D distributions of microbiota within entire tumors largely unexplored. Here, a modified iDISCO-CUBIC tissue clearing and D-amino acid microbiome labeling-based (miCDaL) strategy are proposed, that integrates microbiota in situ labeling, tissue clearing, and whole-mount tissue imaging to enable 3D visualization of indigenous intratumor microbiota. Leveraging whole-mount spatial resolution and centimeter-scale imaging depth, the 3D biogeography of microbiota is successfully charted across various tumors at different developmental stages, providing quantitative spatial insights in relation to host tumors. By incorporating an immunostaining protocol, 3D imaging of the immunologic microenvironment is achieved in both murine and human mammary tumors that is previously assumed to be bacteria-free. Notably, immune infiltrates, including T cells and NK cells, and tertiary lymphoid structures are conspicuously absent in bacteria-colonized regions. This 3D imaging strategy for mapping Indigenous intratumor microbiota offers valuable insights into host-microbiota interactions.

Chemically Stable Diazo Peptides as Selective Probes of Cysteine Proteases in Living Cells.

Diazo peptides have been described earlier, however, due to their high reactivity have not been broadly used until today. Here, we report the preparation, properties, and applications of chemically stable internal diazo peptides. Peptidyl phosphoranylidene-esters and amides were found to react with triflyl azide primarily to novel 3,4-disubstituted triazolyl-peptides. Nonaflyl azide instead furnished diazo peptides, which are chemically stable from pH 1-14 as amides and from pH 1-8 as esters. Thus, diazo peptides prepared by solid phase peptide synthesis were stable to final deprotection with 95% trifluoroacetic acid. Diazo peptides with the recognition sequence of caspase-3 were identified as specific, covalent, and irreversible inhibitors of this enzyme at low nanomolar concentrations. A fluorescent diazo peptide entered living cells enabling microscopic imaging and quantification of apoptotic cells via flow cytometry. Thus, internal diazo peptides constitute a novel class of activity-based probes and enzyme inhibitors useful in chemical biology and medicinal chemistry.

Protocol for protein modification using oxalyl thioester-mediated chemoselective ligation.

The development of fast ligation chemistries for the site-specific modification of proteins has become a major focus in chemical biology. We describe steps for preparing an oxalyl thioester precursor in the form of an N-oxalyl perhydro-1,2,5-dithiazepine handle, i.e., the oxoSEA group, and incorporating it into a peptide modifier using solid phase peptide synthesis. We then detail procedures for its application for the modification of an N-terminal Cys-containing B1 domain of the streptococcal G protein using the native chemical ligation. For complete details on the use and execution of this protocol, please refer to Snella et al.1.

Dual-locked fluorescent probes for precise diagnosis and targeted treatment of tumors.

Cancer continues to pose a significant threat to global health, with its high mortality rates largely attributable to delayed diagnosis and non-specific treatments. Early and accurate diagnosis is crucial, yet it remains challenging due to the subtle and often undetectable early molecular changes. Traditional single-target fluorescent probes often fail to accurately identify cancer cells, relying solely on single biomarkers and consequently leading to high rates of false positives and inadequate specificity. In contrast, dual-locked fluorescent probes represent a breakthrough, designed to enhance diagnostic precision. By requiring the simultaneous presence of two specific tumor-associated biomarkers or microenvironmental conditions, these probes significantly reduce non-specific activations typical of conventional single-analyte probes. This review discusses the structural designs, response mechanisms, and biological applications of dual-locked probes, highlighting their potential in tumor imaging and treatment. Importantly, the review addresses the challenges, and perspectives in this field, offering a comprehensive look at the current state and future potential of dual-locked fluorescent probes in oncology.

A protocol for time-resolved transcriptomics through metabolic labeling and combinatorial indexing.

The snapshot nature of single-cell transcriptomics presents a challenge for studying the dynamics of gene expression. Metabolic labeling, where nascent RNA is labeled with 4-thiouridine (4sU), captures temporal information at the single-cell level, providing greater insight into expression dynamics. Here, we present an optimized, automation-friendly protocol for the metabolic labeling of RNA alongside single-cell RNA sequencing through combinatorial indexing. We describe steps for 4sU labeling, cell fixation and chemical treatment, and automated two-level combinatorial indexing. For complete details on the use and execution of this protocol, please refer to Maizels et al.1.