Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

Abstract

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multiselectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cellimpermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity -displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via colabeling, and assess real-time cell surface receptor traffic via pulsechase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.

Fluorogen activating protein toolset for protein trafficking measurements

“…The dL5** and AM2‐2 FAP tags noncovalently bind MG and TO1 dye derivatives respectively with high affinities (Figures and ). Later on other scFv tags were identified and characterized for binding and activation of dimethylindole red (DIR) and oxazole thiazole‐blue (OTB) fluorogenic dyes, and near‐infrared dyes, SKC1 and SKCi1 (Figure ) . FAP‐dye binding rigidizes the chemical structure, suppressing some bond‐rotations responsible for internal conversion and nonradiative relaxation in the electronic excited state, resulting in increased fluorescence emission.…”

“…Later on other scFv tags were identified and characterized for binding and activation of dimethylindole red (DIR) and oxazole thiazole-blue (OTB) fluorogenic dyes, and near-infrared dyes, SKC1 and SKCi1 ( Figure 5). 23,[28][29][30] FAP-dye binding rigidizes the chemical structure, suppressing some bond-rotations responsible for internal conversion and nonradiative relaxation in the electronic excited state, resulting in increased fluorescence emission. The first synthesized dyes, MG-2p, MG-11p and TO1-2p, possess an hydrophilic diethylene glycol diamine, carrying a positive charge and significant hydration that prevents these dyes from passively crossing the PM and entering the cell.…”

Fluorogen-Activating Proteins: Next-Generation Fluorescence Probes for Biological Research

“…Some examples include fluorescence signal onset and offset, fluorescence color switch, and spatial protein labeling and tracking. 68 ■ CELL-IMPERMEABLE FAP FLUOROGENS Fluorogens constitute the fluorescence modality of FAPs, and similar to conventional chemical fluorophores, these small molecules offer a wide-range of detection properties and colors. The cell impermeable fluorogens permit measurements of cell-surface FAP-tagged proteins without the interference of intracellular and background signals.…”

“…101 A different scFv scaffold exhibited multiselective activation of three different cell impermeant fluorogens, each with a distinct emission channel activation of blue, green, and red. 68 This scFv proved advantageous for fluorescence detection of cell-surface FAPtagged receptors using tricolor labeling. This included real-time signal exchange via fluorogen competition, multichannel detection via colabeling, and real-time cell-surface receptor traffic via pulse-chase.…”

Antibody-Linked Fluorogen-Activating Proteins for Antigen Detection and Cell Ablation

“…A given FAP can activate the fluorescence of multiple chemically-related fluorogens, each with distinct biochemical and spectral properties 4,[10][11][12][13][14][15] . The dL5**/MG pair used here, for instance, emits light in the far red spectrum where tissue autofluorescence and phototoxicity are low 1,5 .…”