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The visualization of drugs in living systems has become key techniques in modern therapeutics. Recent advancements in optical imaging technologies and molecular design strategies have revolutionized drug visualization. At the subcellular level, super-resolution microscopy has allowed exploration of the molecular landscape within individual cells and the cellular response to drugs. Moving beyond subcellular imaging, researchers have integrated multiple modes, like optical near-infrared II imaging, to study the complex spatiotemporal interactions between drugs and their surroundings. By combining these visualization approaches, researchers gain supplementary information on physiological parameters, metabolic activity, and tissue composition, leading to a comprehensive understanding of drug behavior. This review focuses on cutting-edge technologies in drug visualization, particularly fluorescence imaging, and the main types of fluorescent molecules used. Additionally, we discuss current challenges and prospects in targeted drug research, emphasizing the importance of multidisciplinary cooperation in advancing drug visualization. With the integration of advanced imaging technology and molecular design, drug visualization has the potential to redefine our understanding of pharmacology, enabling the analysis of drug micro-dynamics in subcellular environments from new perspectives and deepening pharmacological research to the levels of the cell and organelles.

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Plant peptides communicate by binding to a large family of receptor-like kinases (RLKs), and they share a conserved binding mechanism, which may account for their promiscuous interaction with several RLKs. In order to understand the in vivo binding specificity of the CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptide family in Arabidopsis, we have developed a novel set of CLAVATA3 (CLV3)-based peptide tools. After carefully evaluating the CLE peptide binding characteristics, using solid phase synthesis process, we modified the CLV3 peptide and attached a fluorophore and a photoactivable side group. We observed that the labeled CLV3 shows binding specificity within the CLAVATA1 clade of RLKs while avoiding the distantly related PEP RECEPTOR clade, thus resolving the contradictory results obtained previously by many in vitro methods. Furthermore, we observed that the RLK-bound CLV3 undergoes clathrin-mediated endocytosis and is trafficked to the vacuole via ARA7 (a Rab GTPase)-labeled endosomes. Additionally, modifying CLV3 for light-controlled activation enabled spatial and temporal control over CLE signaling. Hence, our CLV3 macromolecular toolbox can be used to study rapid cell specific down-stream effects. Given the conserved binding properties, in the future our toolbox can also be used as a template to modify other CLE peptides.

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Fluorescent labeling is a broadly utilized approach to assess in vitro and in vivo behavior of biologically active, especially cell-penetrating and antimicrobial peptides. In this communication, far-UV circular dichroism (CD) spectra of penetratin (PEN) fluorophore conjugates reported previously have been re-evaluated. Compared to the intrinsically disordered native peptide, rhodamine B and carboxyfluorescein derivatives of free and membrane-bound PEN exhibit extrinsic CD features. Potential sources of these signals displayed above 220 nm are discussed suggesting the contributions of both intra- and intermolecular chiral exciton coupling mechanisms. Careful evaluation of the CD spectra of fluorophore-labeled peptides is a valuable tool for early detection of labeling-provoked structural alterations which in turn may modify the membrane binding and cellular uptake compared to the unconjugated form.

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Cell-penetrating peptides (CPPs) are versatile tools to deliver various molecules into different cell types. The majority of CPPs are usually represented by linear structures, but numerous recent studies demonstrated cyclization to be an effective strategy leading to favorable biological activities. Here we describe two different methods for the side chain and backbone cyclization of CPPs . Furthermore, we highlight straightforward procedures for the covalent coupling of fluorophores or cytotoxic payloads.

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As analytical glycomics became to prominence, newer and more efficient sample preparation methods are being developed. Albeit, numerous reductive amination based carbohydrate labeling protocols have been reported in the literature, the preferred way to conduct the reaction is in closed vials. Here we report on a novel evaporative labeling protocol with the great advantage of continuously concentrating the reagents during the tagging reaction, therefore accommodating to reach the optimal reagent concentrations for a wide range of glycan structures in a complex mixture. The optimized conditions of the evaporative labeling process minimized sialylation loss, otherwise representing a major issue in reductive amination based carbohydrate tagging. In addition, complete and uniform dispersion of dry samples was obtained by supplementing the low volume labeling mixtures (several microliters) with the addition of extra solvent (e.g., THF). Evaporative labeling is an automation-friendly glycan labeling method, suitable for standard open 96 well plate format operation.

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Single-molecule FRET (smFRET) can visualize conformational dynamics of individual ion channels in lipid bilayers of defined composition. Dynamic and distance measurements from smFRET, combined with single channel recordings, can provide previously unattainable direct mechanistic insights into ion channel function and modulation. smFRET measurements require site-specific fluorophore labeling between two distinct sites, which is a major challenge for multimeric ion channels. This chapter aims to provide a step-by-step protocol: (1) to design concatemeric constructs with only two cysteine residues within a homotetrameric channel; (2) to express, purify, label, and reconstitute channel proteins; (3) to perform smFRET imaging on channel proteins in liposomes with an objective-based Total Internal Reflection (TIRF) microscope; and finally (4) to analyze the FRET distributions and dynamics that reflect the dynamic conformational transitions of ion channels in membranes.

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One of the most frequently used high-resolution glycan analysis methods in the biopharmaceutical and biomedical fields is capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. Glycans are usually labeled by reductive amination with a charged fluorophore containing a primary amine, which reacts with the aldehyde group at the reducing end of the glycan structures. In this reaction, first a Schiff base is formed that is reduced to form a stable conjugate by a hydrogenation reagent, such as sodium cyanoborohydride. In large scale biopharmaceutical applications, such as clone selection for glycoprotein therapeutics, hundreds of reactions are accomplished simultaneously, so the HCN generated in the process poses a safety concern. To alleviate this issue, here we propose catalytic hydrogen transfer from formic acid catalyzed by water-soluble iridium(III)- and ruthenium(II)-phosphine complexes as a novel alternative to hydrogenation. The easily synthesized water-soluble iridium(III) and the ruthenium(II) hydrido complexes showed high catalytic activity in carbohydrate labeling. This procedure is environmentally friendly and reduces the health risks for the industry. Using carbohydrate standards, oligosaccharides released from glycoproteins with highly sialylated (fetuin), high mannose (ribonuclease B) and mixed sialo and neutral (human plasma) N-glycans, we demonstrated similar labeling efficiencies for iridium(III) dihydride to that of the conventionally used sodium cyanoborohydride based reaction. The derivatization reaction time was less than 20min with no bias towards the above mentioned specific glycan structures.

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N-glycan profiling of therapeutic glycoproteins is essential to ensure the activity and efficacy of these promising new-generation drugs. The N-linked glycan moieties of these entities highly affect circulation half-life, immunogenicity and receptor-binding activity as well as physicochemical and thermal stability properties. In addition, more than half of the biopharmaceuticals are glycoproteins representing multibillion dollar worldwide business, further emphasizing the importance of their analysis. In the biomedical field, on the other hand, revealing disease-related glycan structure alterations holds the promise of the discovery of new biomarkers for early diagnostics. Therefore, there is a great demand for widely applicable, high-throughput sample preparation and analysis methods for N-glycan profiling of glycoproteins. One of the newest exciting developments of the field is the magnetic bead based glycoprotein sample preparation technique. A detailed protocol of this method is given in this chapter in conjunction with rapid capillary electrophoresis analysis of the prepared samples by laser induced fluorescence detection (CE-LIF). N-glycans are digested by the endoglycosidase PNGase F and the released carbohydrates are labeled with the charged fluorophore dye of aminopyrenetrisulfonate (APTS). Effective glycan capture by magnetic microparticles enabled fast, easily automated sample preparation both in individual (single vial) and 96-well plate formats, including excess dye removal. Rapid separation of APTS labeled IgG glycans is also shown utilizing an optimized CE-LIF protocol.

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RNA plays a fundamental, ubiquitous role as either substrate or functional component of many large cellular complexes-"molecular machines"-used to maintain and control the readout of genetic information, a functional landscape that we are only beginning to understand. The cellular mechanisms for the spatiotemporal organization of the plethora of RNAs involved in gene expression are particularly poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent mechanistic parameters that govern cellular RNA functions, including those of protein coding messenger RNAs (mRNAs). Progress has been hampered, however, by the scarcity of efficient high-yield methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ T7 RNA polymerase to body label an RNA with a cyanine dye, as well as yeast poly(A) polymerase to strategically place multiple 2'-azido-modifications for subsequent fluorophore labeling either between the body and tail or randomly throughout the tail. Using a combination of biochemical and single-molecule fluorescence microscopy approaches, we demonstrate that both yeast poly(A) polymerase labeling strategies result in fully functional mRNA, whereas protein coding is severely diminished in the case of body labeling.

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Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin-specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of ubiquitin chains when studying how DUBs process these chains.

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Dynamics of actin filaments are regulated by a number of actin-binding proteins. To understand the function of an actin-binding protein, it is necessary to characterize effects of the protein on actin filament dynamics in vitro. This chapter describes basic microscopic methods to visualize fluorescently labeled actin filaments using commonly available fluorescence microscope settings. Direct microscopic observation of actin filaments provides strong evidence for severing or bundling of actin filaments.

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Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid ß (Aß) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aß allows for their detection. The times of half completion of the process (t1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aß fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration.

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