title: 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 multicomponent 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.
authors.affiliation: Departments of Chemistry, ‡Molecular Biology & Biochemistry, and §Pharmaceutical Sciences, University of California , Irvine, California 92697, United States.
authors.forename: Krysten A
authors.initials: KA
authors.lastname: Jones
authors.affiliation: Departments of Chemistry, ‡Molecular Biology & Biochemistry, and §Pharmaceutical Sciences, University of California , Irvine, California 92697, United States.
authors.forename: William B
authors.initials: WB
authors.lastname: Porterfield
authors.affiliation: Departments of Chemistry, ‡Molecular Biology & Biochemistry, and §Pharmaceutical Sciences, University of California , Irvine, California 92697, United States.
authors.forename: Colin M
authors.initials: CM
authors.lastname: Rathbun
authors.affiliation: Departments of Chemistry, ‡Molecular Biology & Biochemistry, and §Pharmaceutical Sciences, University of California , Irvine, California 92697, United States.
authors.forename: David C
authors.initials: DC
authors.lastname: McCutcheon
authors.affiliation: Departments of Chemistry, ‡Molecular Biology & Biochemistry, and §Pharmaceutical Sciences, University of California , Irvine, California 92697, United States.
authors.forename: Miranda A
authors.initials: MA
authors.lastname: Paley
authors.affiliation: Departments of Chemistry, ‡Molecular Biology & Biochemistry, and §Pharmaceutical Sciences, University of California , Irvine, California 92697, United States.
authors.forename: Jennifer A
authors.identifier: 0000-0002-9250-4702
authors.initials: JA
authors.lastname: Prescher
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D048888:Fireflies; D049454:Firefly Luciferin / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D015394:Molecular Structure; D015202:Protein Engineering
chemical_list: D049454:Firefly Luciferin; D008156:Luciferases