基于多路酶活性的杂交探针显示

IF 3.784 3区 化学 Q1 Chemistry
Valerie Cavett, Brian M. Paegel*
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引用次数: 1

摘要

乳剂提供了小型化和并行化高通量筛选的手段,但需要一种强大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,尽管对于识别具有非常特定活性的文库成员是非常理想的。在这里,我们提出了多路探针固定在库珠乳液筛选。在文库珠制备过程中,我们用荧光原位杂交法定量了每珠约106个引物,而乳状PCR每珠仅产生约103个基因拷贝。我们利用未延伸的珠结合引物将互补的探针-寡核苷酸杂联物与文库珠杂交。然后利用探针杂交的蛋白头文库对乳化液体外转录/翻译反应进行编程,并通过FACS对胰蛋白酶和凝乳胰蛋白酶突变文库进行多重活性筛选,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多路复用,并且似乎可扩展到其他酶类和库类型。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Multiplexed Enzyme Activity-Based Probe Display via Hybridization

Multiplexed Enzyme Activity-Based Probe Display via Hybridization

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.

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来源期刊
ACS Combinatorial Science
ACS Combinatorial Science CHEMISTRY, APPLIED-CHEMISTRY, MEDICINAL
自引率
0.00%
发文量
0
审稿时长
1 months
期刊介绍: The Journal of Combinatorial Chemistry has been relaunched as ACS Combinatorial Science under the leadership of new Editor-in-Chief M.G. Finn of The Scripps Research Institute. The journal features an expanded scope and will build upon the legacy of the Journal of Combinatorial Chemistry, a highly cited leader in the field.
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