ArticleACS chemical biology2013
A fluorogenic TMP-tag for high signal-to-background intracellular live cell imaging.
Article in ACS chemical biology, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 33 papers.
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Who cites it
33 citing papers in PubMed, 91 citations in OpenAlex.
- Quantifying protein unfolding kinetics with a high-throughput microfluidic platform.Cell systems · 2026Article
- Peptide Tags for Site-Selective Nonenzymatic Covalent Modification of Proteins.Journal of peptide science : an official publication of the European Peptide Society · 2025Review
- Time-Resolved Analysis of Protein-Protein Ensembles Using a Destabilizing Domain to Map Dynamic Interactions of SARS-CoV-2 nsp15.ACS chemical biology · 2025Article
- Imaging the Binding Between Dasatinib and Its Target Protein in Living Cells Using an SLP Tag System on Intracellular Compartments.International journal of molecular sciences · 2025Article
- Bioisostere-conjugated fluorescent probes for live-cell protein imaging without non-specific organelle accumulation.Chemical science · 2024Article
- Generation of a Recombinant scFv against Deoxycholic Acid and Its Conversion to a Quenchbody for One-Step Immunoassay.Methods and protocols · 2023Article
- Chemical tags and beyond: Live-cell protein labeling technologies for modern optical imaging.Smart molecules : open access · 2023Review
- Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead.Chemical science · 2023Article
- Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP-Tag Technologies.Chembiochem : a European journal of chemical biology · 2020Review
- Genetically Encoded Activators of Small Molecules for Imaging and Drug Delivery.Angewandte Chemie (International ed. in English) · 2020Review
- Fluorogen activating protein toolset for protein trafficking measurements.Traffic (Copenhagen, Denmark) · 2020Review
- Rapid no-wash labeling of PYP-tag proteins with reactive fluorogenic ligands affords stable fluorescent protein conjugates for long-term cell imaging studies.Chemical science · 2020Article
- Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live-Cell Imaging.Chembiochem : a European journal of chemical biology · 2020Article
- Single-Virus Tracking: From Imaging Methodologies to Virological Applications.Chemical reviews · 2020Review
- Covalently Tethered Rhodamine Voltage Reporters for High Speed Functional Imaging in Brain Tissue.Journal of the American Chemical Society · 2020Article
- Development of cyanine probes with dinitrobenzene quencher for rapid fluorogenic protein labelling.Philosophical transactions. Series A, Mathematical, physical, and engineering sciences · 2017Article
- New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR.Archives of biochemistry and biophysics · 2017Review
- Fluorogenic Labeling Strategies for Biological Imaging.International journal of molecular sciences · 2017Review
- Genetically encoded fluorescent tags.Molecular biology of the cell · 2017Article
- The Cation-π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.Biochemistry · 2017Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
Developed to complement the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or nonspecifically bound ligand-fluorophore. We envisioned we could overcome this limit by engineering fluorogenic trimethoprim-based chemical tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR)-tagged protein displaces the quencher. Thus, we began by building a nonfluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here we take the final step and render the covalent TMP-tag fluorogenic. In brief, we designed a trimeric TMP-fluorophore-quencher molecule (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. We present the in vitro experiments showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only our initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins but also less abundant, more dynamic cytoplasmic proteins. These results suggest that the fluorogenic TMP-tag can significantly impact high-resolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.