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

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.

Bioluminescence, photophysical, computational and molecular docking studies of fully conformationally restricted enamine infraluciferin

“…21 This π-extended luciferin provided nIR emission up to 730 nm with mutant CBR2. 11 As the structure retained the 6-hydroxyl group, it was also shown to be sensitive to colour modulation. Later this was capitalised upon by demonstrating nIR dual bioluminescence imaging in mouse models of cancer.…”

Biochemical Analysis Leads to Improved Orthogonal Bioluminescent Tools

“…[5a] While these enzymes represent a notable pair in the field, they still could be optimized further for key biochemical properties. [6] To date, the majority of biochemical studies regarding mutations and their functional impact have focused on color [7] and thermal stability. [8] These studies have been instrumental in both understanding the structure-function relationships of mutations and enabling the rational design of improved probes for applications such as in vitro dual reporter assays.…”

“…[9] Recently, several thoughtful studies have comparatively analyzed combinations of luciferases and luciferins, enabling for an important comparison for scientists choosing a bioimaging tool. [6,10] However, these studies did not examine the role of individual amino acids on the origin of unnatural substrate recognition and do not seek to improve existing tools. While there have been some noteworthy studies on how individual mutations influence modified luciferin recognition, [11] the role of active site residues in modified luciferin substrate selectivity for Fluc is poorly understood.…”

“…Additionally, it is one of a small number of multicomponent luciferase imaging systems shown to enable rapid multicomponent imaging in mice [5a] . While these enzymes represent a notable pair in the field, they still could be optimized further for key biochemical properties [6] …”

“…These studies have been instrumental in both understanding the structure‐function relationships of mutations and enabling the rational design of improved probes for applications such as in vitro dual reporter assays [9] . Recently, several thoughtful studies have comparatively analyzed combinations of luciferases and luciferins, enabling for an important comparison for scientists choosing a bioimaging tool [6,10] . However, these studies did not examine the role of individual amino acids on the origin of unnatural substrate recognition and do not seek to improve existing tools.…”