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 ∼10⁶ primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼10³ 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.

中文翻译：

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通过杂交的基于多重酶活性的探针显示。

乳液提供了使高通量筛选小型化和并行化的方法，但需要一种可靠的方法来定位每个液滴中基于活性的荧光探针。液滴中的多路复用探针是不切实际的，但非常适合识别具有非常特定活性的文库成员。在这里，我们展示了用于乳液筛选的文库珠上的多重探针固定。在文库微珠制备过程中，我们通过荧光原位杂交对每个微珠定量了约 10 ^{6 个}引物，但是乳液 PCR 仅产生了约 10 ^3个引物每个珠子的基因拷贝数。我们利用未延伸的珠子结合引物将互补的探针寡核苷酸杂合物与文库珠子杂交。然后使用探针杂交珠文库对体外转录/翻译反应进行乳液编程，并通过 FACS 进行分析，以对胰蛋白酶和胰凝乳蛋白酶突变体文库进行基于多重活性的筛选，以获得新的蛋白水解特异性。该方法的模块化应该允许高度的探针复用，并且似乎可以扩展到其他酶类和文库类型。