Parallel Screening for Rapid Identification of Orthogonal Bioluminescent Tools,ACS Central Science

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Parallel Screening for Rapid Identification of Orthogonal Bioluminescent Tools
ACS Central Science ( IF 12.7 ) Pub Date : 2017-11-15 00:00:00 , DOI: 10.1021/acscentsci.7b00394
Colin M. Rathbun ₁ , William B. Porterfield ₁ , Krysten A. Jones ₁ , Marian J. Sagoe ₁ , Monique R. Reyes ₁ , Christine T. Hua ₁ , Jennifer A. Prescher ₁

Affiliation

Bioluminescence imaging with luciferase enzymes and luciferin small molecules is a well-established technique for tracking cells and other biological features in rodent models. Despite its popularity, bioluminescence has long been hindered by a lack of distinguishable probes. Here we present a method to rapidly identify new substrate-selective luciferases for multicomponent imaging. Our strategy relies on parallel screening of luciferin analogues with panels of mutant enzymes. The compiled data set is then analyzed in silico to uncover mutually orthogonal sets. Using this approach, we screened 159 mutant enzymes with 12 luciferins. Thousands of orthogonal pairs were revealed with sufficient selectivity for use in biological environments. Over 100 pairs were validated in vitro, and three were applied in cell and animal models. The parallel screening method is both generalizable and scalable and will streamline the search for larger collections of orthogonal probes.

中文翻译：

平行筛选快速鉴定正交生物发光工具

萤光素酶和萤光素小分子的生物发光成像是一种成熟的技术，可用于跟踪啮齿动物模型中的细胞和其他生物学特征。尽管它很受欢迎，但长期以来由于缺乏可区分的探针而阻碍了生物发光。在这里，我们提出了一种快速识别新的多组分成像底物选择性荧光素酶的方法。我们的策略依赖于荧光素类似物与突变酶的平行筛选。然后对经过编译的数据集进行计算机分析，以发现相互正交的集。使用这种方法，我们用12种萤光素筛选了159个突变酶。揭示了成千上万的正交对具有足够的选择性，可用于生物环境。超过100对已通过体外验证，其中三个应用于细胞和动物模型。并行筛选方法既可通用，又可扩展，将简化对更大数量的正交探针的搜索。

更新日期：2017-11-15

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