Hintersteiner Martin, Kimmerlin Thierry, Kalthoff Frank, Stoeckli Markus, Garavel Geraldine, Seifert Jan-Marcus, Meisner Nicole-Claudia, Uhl Volker, Buehler Christof, Weidemann Thomas, Auer Manfred
University of Edinburgh, School of Biological Sciences, The King's Buildings, CH Waddington Building 3.07, Mayfield Road, Edinburgh EH9 3JR, UK.
Chem Biol. 2009 Jul 31;16(7):724-35. doi: 10.1016/j.chembiol.2009.06.011.
Screening of one-bead one-compound libraries by incubating beads with fluorescently labeled target protein requires isolation and structure elucidation of a large number of primary hit beads. However, the potency of the identified ligands is only revealed after time consuming and expensive larger scale resynthesis and testing in solution. Often, many of the resynthesized compounds turn out to be weak target binders in solution due to large differences between surface and solution binding affinities. For an industry style high-throughput screening (HTS) process a high false positive rate is detrimental. We have therefore combined single bead and single molecule/single cell techniques into an integrated HTS process in which the picomole amount of substance contained on one isolated hit bead is sufficient for quality control, structure determination, and precise affinity determination to the target protein in solution.
通过将珠子与荧光标记的靶蛋白孵育来筛选单珠单化合物文库，需要分离和阐明大量初次命中的珠子的结构。然而，所鉴定配体的效力只有在经过耗时且昂贵的大规模重新合成并在溶液中进行测试后才能揭示。由于表面亲和力和溶液亲和力之间存在巨大差异，许多重新合成的化合物在溶液中往往是弱靶标结合剂。对于工业规模的高通量筛选（HTS）过程而言，高假阳性率是有害的。因此，我们将单珠和单分子/单细胞技术结合成一个集成的HTS过程，其中一个分离的命中珠子上所含的皮摩尔量的物质足以用于质量控制、结构测定以及精确测定与溶液中靶蛋白的亲和力。
