Biochemistry. 2021 Mar 2;60(8):563-572. doi: 10.1021/acs.biochem.0c00894. Epub 2021 Feb 18.
Bioluminescence imaging with luciferase-luciferin pairs is routinely used to monitor cellular functions. Multiple targets can be visualized in tandem using luciferases that process unique substrates, but only a handful of such orthogonal probes are known. Multiplexed studies require additional robust, light-emitting molecules. In this work, we report new luciferins for orthogonal imaging that comprise disubstituted cores. These probes were found to be bright emitters with various engineered luciferases. The unique patterns of light output also provided insight into enzyme-substrate interactions necessary for productive emission. Screening studies identified mutant luciferases that could preferentially process the disubstituted analogues, enabling orthogonal imaging with existing bioluminescent reporters. Further mutational analyses revealed the origins of substrate selectivity. Collectively, this work provides insights into luciferase-luciferin features relevant to bioluminescence and expands the number of probes for multicomponent tracking.
荧光素酶-荧光素对的生物发光成像是常规用于监测细胞功能的方法。可以使用能够处理独特底物的荧光酶串联可视化多个靶标，但已知的这种正交探针只有少数几种。多重研究需要额外的稳健、发光分子。在这项工作中，我们报告了新的用于正交成像的包含取代核的荧光素，发现这些探针与各种工程化的荧光酶一起是明亮的发射器。独特的光输出模式也为酶-底物相互作用提供了深入的了解，这些相互作用对于产生有效的发射是必要的。筛选研究确定了可以优先处理取代类似物的突变荧光酶，从而可以使用现有的生物发光报告器进行正交成像。进一步的突变分析揭示了底物选择性的起源。总的来说，这项工作深入了解了与生物发光相关的荧光酶-荧光素特性，并扩展了用于多组分跟踪的探针数量。
