Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications.

Bruce R Branchini,Danielle M Fontaine,Dawn Kohrt,Tara L Southworth,Benjamin R Fort,Brian P Huta,Allison R Racela,Aldo Roda

doi:10.3390/ijms23052451

Abstract

Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH2 and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with luc genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI.

Highlights

Bioluminescence (BL), the emission of visible light by living organisms, is widely distributed in nature and has piqued the curiosity of humans from ancient times to the present
We have provided a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH2 and nine substrate analogs including NH2 -QLH2 and OH-QLH2 that were disclosed here
In predicting based non-invasive animal imaging (BLI) performance; (3) the incredible ~160 nm range of red-shifted BL emission maxima catalyzed by Luc2; (4) further documentation of the

Summary

Introduction

Bioluminescence (BL), the emission of visible light by living organisms, is widely distributed in nature and has piqued the curiosity of humans from ancient times to the present. One commonly observed example of BL is that of the beetles including the widely studied North American firefly Photinus pyralis. As is the case for all characterized bioluminescent beetles, P. pyralis produces light from an enzyme (luciferase, Luc)-catalyzed reaction of a substrate (luciferin, LH2 ) requiring Mg-ATP and molecular oxygen. While P. pyralis Luc (often referred to as PpyWT and Fluc) normally produces yellow-green light (λmax ~560 nm) with LH2 , Luc mutants and various wild-type enzymes can produce emissions with maxima ranging from ~535 nm to ~630 nm [4,5,6,7].

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