Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging

Giorgia Zambito; Mary P Hall; Monika G Wood; Natasa Gaspar; Yanto Ridwan; Fabio F Stellari; Ce Shi; Thomas A Kirkland; Lance P Encell; Clemens Löwik; Laura Mezzanotte

doi:10.1016/j.isci.2020.101986

Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging

iScience. 2020 Dec 26;24(1):101986. doi: 10.1016/j.isci.2020.101986. eCollection 2021 Jan 22.

Authors

Giorgia Zambito^{1

2

3}, Mary P Hall⁴, Monika G Wood⁴, Natasa Gaspar^{1

2

5}, Yanto Ridwan^{1

2}, Fabio F Stellari⁶, Ce Shi⁷, Thomas A Kirkland⁷, Lance P Encell⁴, Clemens Löwik^{1

2

8}, Laura Mezzanotte^{1

2}

Affiliations

¹ Erasmus Medical Center, Radiology and Nuclear Medicine, Rotterdam, 3015 CE, the Netherlands.
² Erasmus Medical Center, Molecular Genetics, Rotterdam, 3015 CE, the Netherlands.
³ Medres Medical Research GmBH, Cologne 50931, Germany.
⁴ Promega Corporation, Madison, WI 53711, USA.
⁵ Percuros B.V., Leiden, 1333 CL, the Netherlands.
⁶ Chiesi Farmaceutici, Parma, 43122, Italy.
⁷ Promega Biosciences Incorporated, San Luis Obispo, CA 93401, USA.
⁸ CHUV Department of Oncology, University of Lausanne, 461011 Lausanne, Switzerland.

Abstract

For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH₂-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate.

Keywords: Biological Services; Biophysics; Optical Imaging.