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Laccase-Mediated Catalyzed Fluorescent Reporter Deposition for Live-Cell Imaging
Brandon T Cisneros1, Neal K Devaraj1
1Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA, 92037, USA.
Insights
This study introduces a novel method for catalyzed reporter deposition (CARD) using laccase, eliminating the need for hydrogen peroxide. This advance enables enhanced fluorescence labeling for live-cell microscopy without sample perturbation.
Area of Science:
- Biochemistry
- Microscopy
- Cell Biology
Background:
- Catalyzed reporter deposition (CARD) is a key microscopy technique for biological sample labeling.
- Conventional CARD methods often use horseradish peroxidase, requiring hydrogen peroxide that can disrupt live-cell imaging.
- There is a need for CARD methods compatible with live-cell microscopy.
Purpose of the Study:
- To develop an alternative CARD method using laccase that bypasses the need for exogenous hydrogen peroxide.
- To demonstrate the feasibility of laccase-based CARD for biological sample labeling.
- To validate the application of this new method in live-cell imaging.
Main Methods:
- Utilized a laccase enzyme, an oxidative enzyme that reduces molecular oxygen, for CARD.
- Employed a fluorescently labeled ferulic acid derivative as a reporter substrate for nontargeted covalent labeling of bovine serum albumin.
- Performed live-cell CARD using an antibody-conjugated laccase targeting a surface-bound molecule.
Main Results:
- Successfully demonstrated proof-of-concept for laccase-mediated CARD.
- Achieved nontargeted covalent labeling of proteins with a fluorescent reporter.
- Showcased successful application in live-cell CARD, producing amplified fluorescence signals.
- Confirmed that the laccase method does not require exogenous hydrogen peroxide.
Conclusions:
- Laccase-based CARD offers a viable, hydrogen peroxide-free alternative for amplified fluorescence labeling.
- This method is suitable for live-cell microscopy, minimizing sample perturbation.
- The developed technique expands the toolkit for advanced biological imaging and analysis.
Abstract:
Catalyzed reporter deposition (CARD) is a widely established method for labeling biological samples analyzed using microscopy. Horseradish peroxidase, commonly used in CARD to amplify reporter signals, requires the addition of hydrogen peroxide, which may perturb samples used in live-cell microscopy. Herein we describe an alternative method of performing CARD using a laccase enzyme, which does not require exogenous hydrogen peroxide. Laccase is an oxidative enzyme which can carry out single-electron oxidations of phenols and related compounds by reducing molecular oxygen. We demonstrate proof-of-concept for this technique through the nontargeted covalent labeling of bovine serum albumin using a fluorescently labeled ferulic acid derivative as the laccase reporter substrate. We further demonstrate the viability of this approach by performing live-cell CARD with an antibody-conjugated laccase against a surface-bound target. CARD using laccase produces an amplified fluorescence signal by labeling cells without the need for exogenous hydrogen peroxide.
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