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Molecular Imaging of Viable Cancer Cells
Published in Shoogo Ueno, Bioimaging, 2020
Intramolecular spirocyclization has been utilized as a fluorescence switching mechanism since the 1960s, as exemplified by fluorescein-based probes such as fluorescein diacetate (FDA), fluorescein di-β-galactoside (FDG), and fluorescein diphosphate (FDP).9 These probes, which have two reactive sites in the molecule, exist in the colorless and non-fluorescent spirolactone form in which the π-conjugation of the fluorophore scaffold is broken, but are converted to the colored and fluorescent xanthene form upon reaction with esterase, β-galactosidase, or phosphatase, respectively. Rhodamine 110 has also been used to prepare activatable fluorescence probes for peptidases, such as serine proteases and caspases, by incorporating a peptide substrate at the amino groups of Rhodamine 110.27 More recently, it was reported that silicon rhodamine derivatives with a 2ʹ-carboxylic acid moiety tend to show a turn-on fluorescence property upon binding to tag proteins such as SNAP-tag or Halo-tag.28 These derivatives exist in the colorless and non-fluorescent spirolactone form, but the ring opens when they bind to the protein surface, and they become fluorescent, providing a wash-free staining procedure for target structures. Rhodamine spiroamide, in which the carboxylic acid at the 2ʹ position of regular rhodamines is converted to amide, has also been utilized as a scaffold of activatable probes for detecting metal ions, and more recently for super-resolution imaging.
Non-VLPs
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Carrocci and Hoskins (2014) developed novel small molecule-based RNA tags by fusion of the MS2 coat to either the E. coli dihydrofolate reductase (MS2-eDHFR) or the so-called SNAP tag (MS2-SNAP), where the SNAP is a domain of human O6-alkylguanine-DNA alkyltransferase that can covalently couple to benzylguanine and its derivatives. These two fusions were used to image RNAs in live S. cerevisiae cells with a diverse range of fluorophores. The use of MS2-eDHFR or MS2-SNAP fusions significantly expands the repertoire of fluorophores available for imaging RNAs in live cells. The eDHFR tag tightly bound fluorescent analogs of trimethoprim, while the SNAP tag reacted with fluorescent benzyl guanine or benzylchloropyrimidine derivatives to form a covalent adduct to the protein. The reporter mRNA in this case encoded six copies of the MS2 stem-loop (Carrocci and Hoskins 2014).
The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Rosa Merlo, Diego Caprioglio, Michele Cillo, Anna Valenti, Rosanna Mattossovich, Castrese Morrone, Alberto Massarotti, Franca Rossi, Riccardo Miggiano, Antonio Leonardi, Alberto Minassi, Giuseppe Perugino
One of the major applications of the SNAP-tag® technology concerns the field of cell biology, where detecting fluorescent-tagged-POIs in living cells represents an important tool to study protein functions and locations53. To test our chemo-enzymatic approach, we first investigated the permeability of BGSN3. Lysates of HEK293T cells pre-treated with BGNS3 were then incubated with the SVG substrate: the absence of any fluorescent signal by gel-imaging only in BG-azide treated lysates demonstrated that the internalisation of BGSN3 was fast (ca. 30 min; Figure 6, lane 3) and at concentrations comparable with commercial cell biology BG-substrates (in the range of <5 µM; Figure 6, lane 8). Preliminary experiments by FACS analysis confirmed that the in vivo cycloaddition between BGSN3 and the BDP-FL DBCO occurred (Figure S7(A)). This was also confirmed for E. coli bacterial cells (Figure S7(B), lane 2).
Current strategies for the discovery and bioconjugation of smaller, targetable drug conjugates tailored for solid tumor therapy
Published in Expert Opinion on Drug Discovery, 2021
Mahendra P. Deonarain, Gokhan Yahioglu
The chemistry around the bioconjugation of earlier small formats such as Fab fragments tend to reflect those of whole antibodies due to the multiple native sites available. In early examples native lysine residues were targeted using activated ester chemistry to produce a heterogenous DAR 1. Lysine modification of Fab fragments are now rarely used and have been superseded by site-specific and bio-orthogonal methods where the appropriate reactive group can be engineered through the insertion or substitution of amino acids and the protein expressed either bacterially or in yeast. The most common approaches involve the replacement of single native residues with cysteines providing reactive thiol groups for classic maleimide conjugation chemistry. The judicious incorporation of two cysteines into the framework, very easy for fragments, facilitates disulfide re-bridging with second-generation maleimides but also opens up the ability to incorporate orthogonal handles like alkynes/azides into next-generation maleimides (NGM) [17] or a pyridazinedione carrying two different orthogonal ‘clickable’ handles permitting dual functionalisation [18]. As the size of the antibody format decreases, reactive cysteines become the dominant technology for bioconjugation along with the insertion of unnatural amino acids to enable site-specific ‘click’ chemistry and peptide tags for site-specific enzymatic conjugation catalyzed by sortases [35]. These unique conjugation sites result in conjugates with defined DAR 1. The SNAP-tag technology is also a promising strategy to produce defined and homogeneous DAR 1 conjugates, enabling the rapid and specific conjugation with benzylguanine modified molecules with fewer chemical manipulations and potentially easier processing [38].
Targets for MAbs: innovative approaches for their discovery & validation, LabEx MAbImprove 6th antibody industrial symposium, June 25-26, 2018, Montpellier, France
Published in mAbs, 2019
Pierre Martineau, Hervé Watier, Andre Pèlegrin, Andrei Turtoi
Dr. Eric Trinquet (CisBio) delivered the next talk on innovative fluorescent technologies to identify and characterize biologics. He highlighted the HTRF technology based on FRET and proprietary fluorescent donors that have long lifetime in fluorescence and time-resolved detections to remove the background. The tag-lite platform combines HTRF and SNAP-tag labeling technology. SNAP-tag involves site-specific covalent labeling of receptors at the surface of living cells. Using this technology, they have developed a wide range of binding assays.10 This technology can be used to screen for binders from phage display libraries of mAbs.