Orthogonal Luciferase–Luciferin Pairs for Bioluminescence Imaging

Abstract

Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate, and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate “hits” were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multi-component imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

Expanding the Chemical Space of Biocompatible Fluorophores: Nanohoops in Cells

“…[1][2][3] The power of such imaging methods has led to increased interest in identifying new types of dyes, opticallyactive materials, and nanoparticles that have enhanced photophysical properties suitable for multimodal, multiplexed, and super-resolution imaging. [4][5][6][7][8][9][10][11][12][13][14] Because fluorophores play such a critical role in understanding biological processes, it is somewhat surprising that most advances in small molecule dye technology today rely on structural modifications of scaffolds discovered over a century ago. [15] For example, the robust Janelia FluorÒ and some AlexaFluorÒ dyes are structurally modified versions of rhodamine scaffolds discovered 130 years ago.…”

Advances in bioluminescence imaging: new probes from old recipes

“…In our own lab, we synthesized dozens of chemically distinct luciferins and screened them against a panel of Fluc mutants. [32,33] A computer algorithm was used to identify orthogonal enzyme-substrate pairs. Substrate selectivity was maintained in both mammalian cells and in mouse models, enabling multi-cellular imaging in vivo (Figure 3B).…”

“…Modular coupling reactions to outfit D-luciferin with diverse steric modifications have been reported. [32,40*] Ring-closing metathesis and carbene insertions have also been used to produce a series of conformationally restricted and pi-extended coelenterazines. [41–43] Many these probes exhibited red-shifted emission or other desirable photophysical properties.…”

Bioluminescent Probes for Imaging Biology beyond the Culture Dish

“…C) Substrate resolution was achieved in mouse DB7 cells using 4′ and 7′-modified luciferins and mutant luciferases identified from screening. Adapted with permission from ref 30 .…”