Department of Pharmaceutical Sciences, University of California, Irvine, California 92617, United States.
Departments of Chemistry & Biomedical Engineering, University of California, Irvine, California 92617, United States.
ACS Comb Sci. 2020 Nov 9;22(11):579-585. doi: 10.1021/acscombsci.0c00116. Epub 2020 Sep 2.
Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼10 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼10 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types.
乳液提供了将高通量筛选微型化和平行化的手段，但需要一种稳健的方法将基于活性的荧光探针定位在每个液滴中。尽管在液滴中对探针进行多重化非常理想，可以识别具有非常特定活性的文库成员，但这是不切实际的。在这里，我们提出了用于乳液筛选的文库珠上的多重探针固定化。在文库珠制备过程中，我们通过荧光原位杂交定量了每个珠上约 10 个引物，但乳液 PCR 每个珠仅产生约 10 个基因拷贝。我们利用未延伸的珠结合引物，使互补探针 - 寡核苷酸异质偶联物与文库珠杂交。然后将探针杂交的珠文库用于编程乳液体外转录/翻译反应，并通过流式细胞术进行分析，以对胰蛋白酶和糜蛋白酶突变文库进行基于活性的新型蛋白水解特异性的多重活性筛选。该方法的模块化应该允许高度的探针多重化，并且似乎可扩展到其他酶类和文库类型。
