Polarization-Activated Droplet Sorting

项目总结/摘要 分子识别几乎涉及生物医学研究的各个方面，从发现新的 从选择性调节靶点功能的治疗先导物到开发新的诊断工具。第一 珠单化合物（OBOC）组合文库合成和筛选具有来源和 廉价且有效地发现这样的配体。然而，该过程受到假阳性选择的困扰 显示配体的珠粒，所述配体一旦从珠粒释放就不重现它们的栓系结合行为 面因此，所有组合文库筛选必须结合命中再合成、纯化和纯化。 用于验证的溶液相结合测定，使得假阳性的调查资源密集， 否定组合文库合成和筛选的效率。直接筛选 用于溶液相靶结合的每个文库成员将减轻上述负担，但是这种 技术还不存在。该提案将探索偏振激活液滴分选（PADS）， 将能够进行超小型化的皮升规模的溶液相荧光偏振（FP） 在初步筛选期间对每个文库成员进行分析。DNA编码的组合文库珠将被 用荧光标记的配体制备，所述配体通过可光裂解的接头连接到珠上，分布到靶- 含有微流体液滴，在流动中从珠粒光化学裂解，在液滴中测定 通过激光诱导的FP结合，并且如果超过FP阈值则进行分选。港口及机场发展策略的审核工作包括 检查各种阳性对照真实配体及其已知靶（例如，链霉亲和素/生物素， 胃蛋白酶抑制剂A/HIV-1蛋白酶），并继续进行在发展中发现的假阳性的已知病例。 筛选HIV-1 gp 120/CD 4-IG复合物的CCR 5模拟配体。港口及机场发展策略的评估工作，会考虑该策略能否 在这些液滴规模的溶液相结合中区分真实配体与已知的假阳性 测定。PADS的成功开发将允许筛选不同的（>105个成员）小分子 用于真实结合的OBOC文库，需要可忽略量的靶。港口及机场发展策略会将效率 DNA编码的组合文库合成和小型化筛选的优点，以提供高度 可扩展的平台，用于发现新的分子工具和治疗线索。

Project Summary / Abstract Molecular recognition touches virtually every aspect of biomedical research, from the discovery of new therapeutic leads that selectively modulate a target's function to the development of new diagnostic tools. One- bead-one-compound (OBOC) combinatorial library synthesis and screening has the potential to source and discover such ligands cheaply and efficiently. However, the process is plagued by the selection of false positive beads displaying ligands that do not recapitulate their tethered binding behavior once liberated from the bead surface. All combinatorial library screening must therefore incorporate hit re-synthesis, purification, and solution-phase binding assays for validation, making the investigation of false positives resource intensive to the point of negating the efficiency of the combinatorial library synthesis and screening. Directly screening each library member for solution-phase target binding would alleviate the burdens described above, but such technology does not yet exist. This proposal will explore polarization-activated droplet sorting (PADS), which will be capable of performing ultra-miniaturized picoliter-scale solution-phase fluorescence polarization (FP) assays on each library member during primary screening. DNA-encoded combinatorial library beads will be prepared with fluorescently-labeled ligand linked to the bead via photocleavable linker, distributed into target- containing microfluidic droplets, photochemically cleaved from the bead in flow, assayed in droplets for binding by laser-induced FP, and sorted if the FP threshold is exceeded. Validation of PADS will involve examining various positive control authentic ligands and their known targets (e.g., streptavidin/biotin, pepstatin A/HIV-1 protease) and proceed to a known case of a false positive discovered in the development of a screen for CCR5-mimetic ligands of the HIV-1 gp120/CD4-Ig complex. PADS will be assessed for its ability to differentiate the authentic ligand from the known false positive in these droplet-scale solution-phase binding assays. The successful development of PADS will allow screening of diverse (>105 members) small molecule OBOC libraries for authentic binding, requiring negligible amounts of target. PADS will integrate the efficiency advantages of DNA-encoded combinatorial library synthesis and miniaturized screening to provide a highly scalable platform for the discovery of new molecular tools and therapeutic leads.