Prenylation (isopentenylation), a key post-transcriptional modification with a hydrophobic prenyl group onto the biomacromolecules such as RNA and proteins, influences their localization and function. Prenyltransferases mediate this process, while cytokinin oxidases degrade the prenylated adenosine in plants. This review summarizes current progress in detecting prenylation modifications in RNA across species and their effects on protein synthesis. Advanced methods have been developed to label and study these modifications in vitro and in vivo, despite challenges posed by the inert chemical properties of prenyl groups. Continued advancements in bioorthogonal chemistry promise new tools for understanding the precise biological functions of prenylated RNA modifications and other related proteins.

Migrasomes are a novel type of cell organelle that form on the retraction fibers at the rear of migrating cells. In recent years, numerous studies have unveiled the mechanisms of migrasome formation and have highlighted significant roles of migrasomes in both physiological and pathological processes. Building upon the strategies outlined in published works and our own research experiences, we have compiled a comprehensive set of protocols for observing migrasomes. These step-by-step instructions encompass various aspects such as cell culture, labeling, imaging, in vitro reconstitution, and statistical analysis. We believe that these protocols serve as a valuable resource for researchers exploring migrasome biology.

Double-stranded RNA (dsRNA) is the replicate intermediate of all RNA viruses, and is also recognized by their host cells as a virus-invading molecule signal. Analysis of the localization and dynamic of virus-induced dsRNA can reveal crucial information concerning the molecular mechanism of virus replication and host responses to viral infection. In this chapter, we provide an easy and efficient protocol called dsRNA binding-dependent fluorescence complementation (dRBFC) assay for labeling the dsRNAs in living plant cells using two different plant RNA viruses, namely potato virus X and turnip mosaic virus. Moreover, both YFP- and mRFP-based dRBFC plasmids are available for the flexibility of experiment design.

The development of organoid research has raised new requirements for this methodology. In a previous study, we demonstrated that an emerging protocol achieved the collection, loading, and programmed immunolabeling of mouse intestinal organoids based on a strainer platform. To uncover the applied potential of this novel methodology on organoids from other species, the strainer platform was utilized to characterize the porcine epidemic diarrhea virus (PEDV)-infected porcine intestinal organoid model. Based on a previous study, some steps were changed to improve the efficiency of the assay by simplifying the reagent addition procedure. In addition, we redefined the range of strainer sizes on porcine intestinal organoids, showing that strainers with pore sizes of 40 and 70 μm matched the above protocol well. Notably, the strainer platform was successfully used to label viral proteins, laying the foundation for its application in the visualization of viral infection models. In summary, the potential of the strainer platform for organoid technology was explored further. More extensive exploration of this platform will contribute to the development of organoid technology.

β-Glucuronidase (GUSB) plays an important role in human physiological and pathological activities. The activity level of GUSB is closely related to human health and diseases. It is imperative to detect the activity of GUSB for related disease diagnosis and treatment. However, exactly evaluating the activity of GUSB in complicated biological system remains a challenge. In this study, we developed photoaffinity-based probes (AfBPs) equipped with photosensitive benzophenone group for labeling active GUSB. Through molecule docking, we predicted the binding model of the AfBPs and GUSB, and the obtained results suggested thermodynamically favorable binding. The AfBPs indicated high efficiency and showed dose-/time-dependent labeling of Escherichia coli (E. coli) GUSB. The application of AfBPs toward GUSB provides a powerful tool to study the activity of target enzymes and contributes to huge potential of enzyme inhibitor discovery and biomedical diagnostics.

Intestinal organoids have emerged as powerful model systems for studying the complex structure and function of the intestine. However, there is a lack of widely applicable methods for the collection, labeling, and imaging of intestinal organoids. In this study, we developed a novel method for loading and labeling intestinal organoids, a method that efficiently collects the organoids and facilitates imaging of their three-dimensional (3D) structure. Based on this strainer platform, mouse intestinal organoids were adequately collected and immobilized, facilitating the immunolabeling workflow to target proteins of the organoids. After evaluation, the strainer size of 40 μm was considered to be more conducive to the collection and labeling of mouse intestinal organoids. More extensive research on organoids of multiple types and species origins will contribute to broadening the applicability of the methodology. Overall, our study proposes an innovative workflow for loading and analyzing intestinal organoids. The combination of a strainer-based collection method, fluorescent labeling, and 3D reconstruction provides valuable insights into the organization and complexity of these tissue models, thereby offering new avenues for investigating intestinal development, disease modeling, and drug discovery.

Peptides can be used as effective molecular tool for covalent modification of proteins and play important roles in ligand directed covalent modification. Tyr-selective protein modifications exert a profound impact on protein functionality. Here, we developed a general strategy that involves nucleophilic addition of alkyne for tyrosine modification. The terminal alkyne of propargyl sulfonium is motivated by the sulfonium center to react with phenolic hydroxyl. This approach provides a straightforward method for tyrosine modification due to its high yield in aqueous solution at physiological temperature. In addition, cyclic peptides could be obtained via adjusting pH to 8.0 from peptides consisting of tyrosine and methionine modified by propargyl bromide, and the resulting cyclic peptides are proved to have better stability, excellent 2-mercaptopyridine resistance and improved cellular uptakes. Furthermore, molecules made from the propargylated sulfonium have the potential to be used as warheads against tyrosine containing biomolecules. Collectively, we develop a direct and uncomplicated technique for modifying tyrosine residues, the strategy concerned can be widely utilized to construct stable peptides and biomolecules imaging.

To explore the new iodine labeling method of nanomaterials, graphene oxide (GO) was labeled by 131I with AgI nanoparticles. As a control, GO was also labeled by 131I with chloramine-T method. The stability of the two 131I labeling materials, viz. [131I]AgI-GO and [131I]I-GO was evaluated. The results show that [131I]AgI-GO is very stable in inorganic environment such as PBS and saline. However, it is not stable enough in serum. The instability of [131I]AgI-GO in serum can be attributed to the higher affinity of Ag to S of thiol group in cysteine than iodine ions and much more chance of interaction between thiol group and [131I]AgI nanoparticles on two-dimensional GO than in three-dimensional nanomaterials.

The common fluorescent conjugated materials present weak or quenching luminescent phenomena in the solid or aggregate state (ACQ), which limits their applications in medicine and biology. In the last two decades, certain materials, named aggregation-induced emission (AIE) fluorescent materials, have exhibited strong luminescent properties in the aggregate state, which can overcome the ACQ phenomenon. Due to their intrinsic properties, the AIE materials have been successfully used in biolabeling, where they can not only detect the species of ions and their concentrations in organisms, but can also monitor the organisms\' physiological activity. In addition, these kinds of materials often present non-biological toxicity. Thus, AIE materials have become some of the most popular biofluorescent probe materials and are attracting more and more attention. This field is still in its early infancy, and several open challenges urgently need to be addressed, such as the materials\' biocompatibility, metabolism, and so on. Designing a high-performance AIE material for biofluorescent probes is still challenging. In this review, based on the molecular design concept, various AIE materials with functional groups in the biofluorescent probes are introduced, including tetrastyrene materials, distilbene anthracene materials, triphenylamine materials, and hexaphenylsilole materials. In addition, according to the molecular system design strategy, the donor-acceptor (D-A) system and hydrogen-bonding AIE materials used as biofluorescent probes are reviewed. Finally, the biofluorescent probe design concept and potential evolution trends are discussed. The final goal is to outline a theoretical scaffold for the design of high-performance AIE biofluorescent probes that can at the same time further the development of the applications of AIE-based biofluorescent probes.

A major concern with antibody labeling is the decreased antigen affinity binding capacity of antibodies, owing mainly to the randomly oriented binding of the marker. Herein, a universal approach for site-specific photocrosslinking of quantum dots (QDs) to the Fc-terminal of antibodies was investigated utilizing antibody Fc-terminal affinity proteins. Results showed that the QDs only bound to the heavy chain of the antibody. Further comparative tests confirmed that the site-specific directed labeling approach maximizes the retention of the antigen-binding capacity of the natural antibody. Compared with the commonly employed random orientation labeling approach, the directional labeling approach allows the labeled antibody showed 6 times greater binding affinity to antigen. QDs-labeled monoclonal antibodies were applied to fluorescent immunochromatographic test strips for the detection of shrimp tropomyosin (TM). The established procedure has a detection limit of 0.054 μg/mL. Thus, the site-specific labeling approach significantly improves the antigen binding capacity of the labeled antibody.