Renilla Luciferase Variants with Green-Peaked Emission Spectra for Improved Imaging in Living Subjects

Renilla luciferase (RLuc) is commonly used as a reporter gene either on its own, or in conjunction with firefly and other beetle luciferases. Its use in small animal imaging, however, has been hampered by its 481 nm peaked emission spectrum as blue wavelengths are strongly attenuated in biological tissues. To overcome this difficulty, we focused on red-shifting RLuc through a combination of semi-rational and random mutagenesis yielding variants with bathochromic shifts of up to 66 nm (547 nm peak). Analysis of the most promising variant demonstrated it was 6-fold brighter than RLuc, and its green emission spectrum (535 nm peak) generated a further ∼3-fold improvement in photon transport at depths of 1-2 mm of animal tissue. This is a significant improvement for bioluminescence imaging over previous RLuc variants, and should greatly aid the use of Renilla luciferase as an in vivo reporter gene.