On-bead screening of combinatorial libraries: reduction of nonspecific binding by decreasing surface ligand density.
On-bead screening of combinatorial libraries: reduction of nonspecific binding by decreasing surface ligand density.
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DOI：
10.1021/cc9000168
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发表时间：
2009-07
影响因子：
--
通讯作者：
Pei, Dehua
中科院分区：
文献类型：
--
作者：
Chen, Xianwen;Tan, Pauline H.;Zhang, Yanyan;Pei, Dehua
单头单化合物（OBOC）文库的头上筛选为快速鉴定针对分子或细胞靶标的活性化合物提供了一种强有力的方法。然而，珠上筛选容易受到非特异性结合的干扰，这导致筛选数据有偏差和假阳性。在这项工作中，我们发现非特异性结合的一个主要来源来自库珠上的高配体负载，这允许大分子靶标（例如，蛋白质）同时与珠表面的多个配体相互作用。为了解决这个问题，我们在空间分离的TentaGel微珠上合成了一个含磷酸酪氨酸（pY）的肽库，其特征是在头表面减少了10倍的肽负载，而在头内部则是正常的肽负载。该文库针对10个Src同源2 （SH2）结构域进行筛选，包括Csk和Fyn激酶以及接头蛋白SLAP，并成功鉴定出每个结构域的特异性识别基序。相比之下，当SH2结构域与含有未改变的（高）配体负载的头表面对照文库进行筛选时，其中6个结构域表现出不同程度的序列偏差，从不同序列相对丰度的轻微扰动到对靶蛋白没有可测量亲和力的假阳性序列的排他选择。这些结果表明，减少微球表面的配体负载是一种简单有效的策略，可以在很大程度上消除非特异性结合的干扰，同时在微球内部保留足够数量的材料用于化合物鉴定。这一发现将进一步扩大OBOC库在生物医学研究中的应用。
On-bead screening of one-bead-one-compound (OBOC) libraries provides a powerful method for the rapid identification of active compounds against molecular or cellular targets. However, on-bead screening is susceptible to interference from nonspecific binding, which results in biased screening data and false positives. In this work, we have found that a major source of nonspecific binding is derived from the high ligand loading on the library beads, which permits a macromolecular target (e.g., a protein) to simultaneously interact with multiple ligands on the bead surface. To circumvent this problem, we have synthesized a phosphotyrosyl (pY)-containing peptide library on spatially segregated TentaGel microbeads, which feature a 10-fold reduced peptide loading on the bead surface but a normal peptide loading in the bead interior. The library was screened against a panel of 10 Src homology 2 (SH2) domains including those of Csk and Fyn kinases and adaptor protein SLAP, and the specific recognition motif(s) was successfully identified for each of the domains. In contrast, when the SH2 domains were screened against a control library that contained unaltered (high) ligand loading at the bead surface, six of them exhibited varying degrees of sequence biases, ranging from minor perturbation in the relative abundance of different sequences to the exclusive selection of false positive sequences that have no measurable affinity to the target protein. These results indicate that reduction of the ligand loading on the bead surface represents a simple, effective strategy to largely eliminate the interference from nonspecific binding, while preserving sufficient amounts of materials in the bead interior for compound identification. This finding should further expand the utility of OBOC libraries in biomedical research.
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影响因子：
4.7
作者：
Aggarwal, S;Harden, JL;Denmeade, SR
通讯作者：
Denmeade, SR
影响因子：
30.5
作者：
Myers, MD;Sosinowski, T;Weiss, A
通讯作者：
Weiss, A
DOI：
10.1073/pnas.0608965103
发表时间：
2006-11-28
影响因子：
11.1
作者：
Dragone, Leonard L.;Myers, Margaret D.;Weiss, Arthur
通讯作者：
Weiss, Arthur
影响因子：
7
作者：
Huang, Haiming;Li, Lei;Li, Shawn S. -C.
通讯作者：
Li, Shawn S. -C.
影响因子：
3.6
作者：
Debenham, SD;Snyder, PW;Toone, EJ
通讯作者：
Toone, EJ