Tiny Titans: Nanobodies as Powerful Tools for TR‐FRET Assay Development

Owing to their exceptional sensitivity and specificity, assays based on time‐resolved Förster resonance energy transfer (TR‐FRET) have become increasingly popular in biomedical research. TR‐FRET assays are highly versatile and compatible with complex biological environments. However, the paucity of...

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Bibliographic Details
Published in:Analysis & sensing 2022-11, Vol.2 (6), p.n/a
Main Authors: Connor Payne, N., Mazitschek, Ralph
Format: Article
Language:English
Subjects:
Adaptation
Antibodies
Assaying
Chemical compounds
Energy transfer
Fluorescence
fluorescent proteins
FRET
immunoassays
luminescence
Modular design
nanobodies
Proteins
Sensitivity
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Summary:Owing to their exceptional sensitivity and specificity, assays based on time‐resolved Förster resonance energy transfer (TR‐FRET) have become increasingly popular in biomedical research. TR‐FRET assays are highly versatile and compatible with complex biological environments. However, the paucity of straightforward strategies for the selective installation of TR‐FRET donor fluorophores on target proteins, which is a critical requirement for most assay designs, has limited the potential and adaptation of this powerful technology. To address this issue, we have developed a versatile toolbox strategy based on CoraFluor‐1‐functionalized nanobodies. The target‐agnostic modular design simplifies the TR‐FRET donor‐labeling of epitope tags, fluorescent proteins and primary antibodies, including their use with endogenous proteins in lysates. Our strategy, which we extend to enable the seamless adaptation of split‐luciferase systems for TR‐FRET approaches, greatly facilitates the development and design of TR‐FRET assays for both target engagement studies and the quantification of proteins of interest with sub‐nanomolar sensitivity. TR‐FRET offers many advantages over other assay modalities. However, installation of the TR‐FRET donor on target proteins can be challenging. CoraFluor‐labeled nanobodies provide a facile and versatile strategy that alleviates this bottleneck. Similarly, LgBiT split‐luciferase can be repurposed as a pseudo‐nanobody, enabling seamless adaptation of TR‐FRET to existing HiBiT luminescence platforms.
ISSN:2629-2742
2629-2742
DOI:10.1002/anse.202200020