Laccase‐Mediated Catalyzed Fluorescent Reporter Deposition for Live‐Cell Imaging

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.

Functionalized polysaccharides improve sensitivity of tyramide/peroxidase proximity labeling assays through electrostatic interactions

“…6,[15][16][17] These methods link enzymatic activity, either directly or through reaction cascades, to the production of highly reactive and fluorescent phenoxyl radical species, which are utilized for labeling purposes. Commonly, an oxidoreductase, such as horseradish peroxidase (HRP), engineered ascorbic acid peroxidase (APEX2), 18,19 laccase, 20 or tyrosinase, 21 is introduced into the labeling reaction to produce the phenoxyl radical by reaction with the by-products of the main enzymatic reaction at the cell surface, after the addition of an enzyme-specific substrate (Figure 1A). The dye-labeled phenoxyl radical then covalently binds to the cell surface through tyrosine moieties, permitting detection.…”