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 Stellari, Ce Shi, Thomas A. Kirkland, Lance P. Encell, Clemens Löwik, Laura Mezzanotte
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.