Polarization-Activated Droplet Sorting

项目摘要/摘要 分子识别几乎触及了生物医学研究的方方面面，从发现新的 有选择地调节靶点功能的治疗导向新诊断工具的开发。一是- 珠一化合物(OBOC)组合文库的合成和筛选具有潜在的来源和 以廉价和高效的方式发现这样的配体。然而，这一过程受到了假阳性选择的困扰 显示配体的珠子，一旦从珠子中解放出来，就不会重述它们的系绳结合行为 浮出水面。因此，所有组合文库筛选都必须包含HIT重新合成、纯化和 用于验证的解决方案阶段绑定分析，使假阳性的调查资源密集到 否定组合文库合成和筛选效率的观点。直接筛选 用于解决方案阶段靶标结合的每个库成员将减轻上述负担，但是 技术还不存在。这项提议将探索极化激活液滴分类(PADS)，它 将能够执行超小型皮升尺度的溶液相荧光偏振(FP) 在初步筛选期间对每个文库成员进行化验。DNA编码的组合库珠将被 用荧光标记的配体通过可光切割的连接物连接到珠子上制备，分布到靶标中- 含有微流控液滴，在流动中以光化学方法从珠子中分离出来，在液滴中进行分析 通过激光诱导FP结合，如果超过FP阈值则进行分类。港口及机场发展策略的验证将包括 检查各种阳性对照的可信配体及其已知靶标(例如，链霉亲和素/生物素， 胃抑素A/HIV-1蛋白酶)，并继续处理在发展过程中发现假阳性的已知病例 筛选HIV-1gp120/CD4-Ig复合体的CCR5模拟配体。将对PAD进行评估，以确定其 在这些液滴尺度的溶液相结合中区分真正的配体和已知的假阳性 化验。PADS的成功开发将允许筛选不同的(105个成员)小分子 OBOC文库进行可信的装订，需要的靶标数量可以忽略不计。PADS将整合效率 DNA编码的组合文库合成和小型化筛选的优点提供了高度的 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.