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Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging† † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1sc03114g

ABSTRACT: Multi-component bioluminescence imaging requires an expanded collection of luciferase–luciferin pairs that emit far-red or near-infrared light. Toward this end, we prepared a new class of luciferins based on a red-shifted coumarin scaffold. These probes (CouLuc-1s) were accessed in a two-step sequence via direct modification of commercial dyes. The bioluminescent properties of the CouLuc-1 analogs were also characterized, and complementary luciferase enzymes were identified using a two-pronged screening strategy. The optimized enzyme-substrate pairs displayed robust photon outputs and emitted a significant portion of near-infrared light. The CouLuc-1 scaffolds are also structurally distinct from existing probes, enabling rapid multi-component imaging. Collectively, this work provides novel bioluminescent tools along with a blueprint for crafting additional fluorophore-derived probes for multiplexed imaging. Near-infrared probes were developed from coumarin-modified luciferins and engineered luciferases, enabling facile multiplexed bioluminescence imaging.

SUBMITTER: Yao Z

PROVIDER: S-EPMC8442684 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bioluminescence is widely used for real-time imaging in living organisms. This technology features a light-emitting reaction between enzymes (luciferases) and small molecule substrates (luciferins). Photons produced from luciferase-luciferin reactions can penetrate through heterogeneous tissue, enabling readouts of physiological processes. Dozens of bioluminescent probes are now available and many are routinely used to monitor cell proliferation, migration, and gene expression patterns in vivo. Despite the ubiquity of bioluminescence, traditional applications have been largely limited to imaging one biological feature at a time. Only a handful of luciferase-luciferin pairs can be easily used in tandem, and most are poorly resolved in living animals. Efforts to develop spectrally distinct reporters have been successful, but multispectral imaging in large organisms remains a formidable challenge due to interference from surrounding tissue. Consequently, a lack of well-resolved probes has precluded multicomponent tracking. An expanded collection of bioluminescent probes would provide insight into processes where multiple cell types drive physiological tasks, including immune function and organ development. We aimed to expand the bioluminescent toolkit by developing substrate-resolved imaging agents. The goal was to generate multiple orthogonal (i.e., noncross-reactive) luciferases that are responsive to unique scaffolds and could be used concurrently in living animals. We adopted a parallel engineering approach to genetically modify luciferases to accept chemically modified luciferins. When the mutants and analogs are combined, light is produced only when complementary enzyme-substrate partners interact. Thus, the pairs can be distinguished based on substrate selectivity, regardless of the color of light emitted. Sequential administration of the luciferins enables the unique luciferases to be illuminated (and thus resolved) within complex environments, including whole organisms. This Account describes our efforts to develop orthogonal bioluminescent probes, crafting custom luciferases (or "biological flashlights") that can selectively process luciferin analogs (or "batteries") to produce light. In the first section, we describe synthetic methods that were key to accessing diverse luciferin architectures. The second section focuses on identifying complementary luciferase enzymes via a combination of mutagenesis and screening. To expedite the search for orthogonal enzymes and substrates, we developed a computational algorithm to sift through large data sets. The third section features examples of the parallel engineering approach. We identified orthogonal enzyme-substrate pairs comprising two different classes of luciferins. The probes were vetted both in cells and whole organisms. This expanded collection of imaging agents is applicable to studies of immune function and other multicomponent processes. The final section of the Account highlights ongoing work toward building better bioluminescent tools. As ever-brighter and more selective probes are developed, the frontiers of what we can "see" in vivo will continue to expand.

Dataset Information

Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging† † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1sc03114g

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