Improved HaloTag Ligand Enables BRET Imaging With NanoLuc

Ovia Margaret Thirukkumaran; Congrong Wang; Nnamdi Joseph Asouzu; Eduard Fron; Susana Rocha; Johan Hofkens; Luke D Lavis; Hideaki Mizuno

doi:10.3389/fchem.2019.00938

Improved HaloTag Ligand Enables BRET Imaging With NanoLuc

Front Chem. 2020 Jan 14:7:938. doi: 10.3389/fchem.2019.00938. eCollection 2019.

Authors

Ovia Margaret Thirukkumaran^{1

2}, Congrong Wang¹, Nnamdi Joseph Asouzu¹, Eduard Fron², Susana Rocha², Johan Hofkens², Luke D Lavis³, Hideaki Mizuno¹

Affiliations

¹ Laboratory for Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Heverlee, Belgium.
² Chem Tech-Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, Belgium.
³ Howard Hughes Medical Institute, Ashburn, VA, United States.

Abstract

Bioluminescence resonance energy transfer (BRET) from an exceptionally bright luciferase, NanoLuc, to a fluorescent HaloTag ligand is gaining momentum to monitor molecular interactions. The recommended use of HaloTag618 ligand for the NanoLuc-HaloTag BRET pair is versatile for ensemble experiments due to their well-separated emission bands. However, this system is not applicable for single-cell BRET imaging because of its low BRET efficiency and in turn weak acceptor signals. Here we explored the unprecedented potential of rhodamine based HaloTag ligands, containing azetidine rings, as BRET acceptors. Through a comprehensive evaluation of various commercial and Janelia Fluor HaloTag ligands for improved BRET efficiency and minimal donor signal bleed-through, we identified JF525 to be the best acceptor for microscopic BRET imaging. We successfully employed BRET imaging with JF525 to monitor the interaction of protein kinase A catalytic and regulatory subunit. Single-cell BRET imaging with HaloTag JF525 can henceforth open doors to comprehend and interpret molecular interactions.

Keywords: BRET imaging; HaloTag; Janelia Fluor dyes; NanoLuc; PKA.