Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging

Mary P Hall,Keith V Wood,Clemens Löwik,Laura Mezzanotte,Ce Shi,Moniek Van’T Root,Monika G Wood,Thomas A Kirkland,Lance P Encell,Ivo Que,Carolyn C Woodroofe,Yanto Ridwan

doi:10.1038/s41467-017-02542-9

Abstract

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system’s peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.

Highlights

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high
It was previously demonstrated that extension of π conjugation in luciferin analogs reduces the HOMO-LUMO energy gap in corresponding oxyluciferins to result in a red-shifted spectrum[11,12,13,14]
In this study we report the design and characterization of two naphthyl-based luciferin analogs and the development of a mutant luciferase enzyme (CBR2) that can efficiently utilize these substrates to produce near infrared (NIR) bioluminescence

Summary

Introduction

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system’s peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Mutagenesis has been used successfully to red-shift the spectral properties of luciferases (utilizing D-LH2 as substrate), but mutants with a significant NIR component to their emission have been elusive[6, 7] This is likely an inherent limitation of the actual photon-emitting species, oxyluciferin[6, 8,9,10]. The utility of these substrates is still limited due to the fact that bioluminescence signals are only enhanced over Luc2/D-LH2 at limited substrate concentrations

Methods

Results

Conclusion