Genome-Inspired Pathway to Aptamer Discovery

 描述(申请人提供)：自1990年推出以来，适体已成为抗体的主流替代品，作为从小分子到大蛋白质甚至整个细胞的靶标的亲和试剂。与抗体相比，它们提供了重要的优势，使它们在广泛的分析和医疗应用中具有吸引力。适配子发现的过程始于构建一个“随机”寡核苷酸文库，该文库通过迭代选择过程，即指数富集型配体的系统进化(SELEX)，减少到少数几个与靶标表现出高亲和力的序列。毫无疑问，适配子一旦被发现，就能兑现他们的承诺。实现其全部潜力的限制因素是，尽管付出了大量的努力和大量的时间和金钱投入，但仍然选择了数量相对较少的适体靶标。这些失败归因于许多因素，包括SELEX过程本身，该过程以繁琐、非标准化和难以完全自动化而臭名昭著。然而，即使这些问题得到解决，SELEX仍然存在一些根本性的困难，这些困难将继续阻碍适体发现方面的进展。这些问题包括组合文库的质量，某些结构基序的表达不足，以及聚合酶链式反应扩增的低效和偏差。这一建议提供了适体发现的范式转变，克服了适体选择的许多障碍。拟议的基因组启发方法逆转了选择，使用人类基因组中的特定DNA序列从天然池中捕获蛋白质，如核蛋白提取物。这种方法利用了亿万年的生物进化产生的基因组DNA序列，这些DNA序列选择性地与蛋白质结合来执行生物功能。具体地说，目标是 目的是识别与乳腺癌基因启动子区域的G-四链(G4)序列特异结合的蛋白质。虽然G4是一个非常成功的适体基序，但在组合文库中表达不足；现有的组合文库仅限于两层G4结构，易于进行低效的PCR扩增。相反，在包括乳腺癌基因启动子区域在内的整个人类基因组中，存在着大量多样性的多层G4结构。拟议的方法挖掘这些以前未被开发的结构，为适体进入新的蛋白质靶点打开了大门。具体目的1是系统地评价所提出的反向选择方法，该方法使用乳腺癌相关人类基因启动子区域的基因组G4形成序列来捕获从培养的乳腺癌和正常乳腺组织细胞系的细胞核中提取的蛋白质。具体目的2是进行染色质免疫沉淀(CHIP)，以确定体外选择的蛋白质是否与活的培养细胞的染色质中包含相应G4形成序列的基因启动子区域结合。这项研究将导致与乳腺癌相关的核调控过程中涉及的蛋白质的适配子，可能包括尚未被认为是SELEX重要靶点的蛋白质。

 DESCRIPTION (provided by applicant): Since their introduction in 1990, aptamers have become an increasingly mainstream alternative to antibodies as affinity reagents for targets ranging from small molecules to large proteins and even whole cells. They offer important advantages over antibodies that make them attractive candidates for wide-ranging analytical and medical applications. The process of aptamer discovery begins with construction of a "random" oligonucleotide library that is reduced through an iterative selection process, Systematic Evolution of Ligands by Exponential Enrichment (SELEX), to a handful of sequences that exhibit high affinity to the target. There is no question that aptamers, once discovered, can live up to their promise. The limiting factor in achieving their full potential is the relatively sall number of protein targets to which aptamers have been selected despite intensive effort and substantial investments of time and money. These failures have been attributed to a number of factors including the SELEX process itself, which is notoriously laborious, non-standardized and difficult to fully automate. Yet even if these problems are solved, there remain fundamental difficulties inherent in SELEX that will continue to hinder progress in aptamer discovery. These include the quality of the combinatorial library, under-representation of certain structural motifs and inefficiencies and bias of PCR amplification. This proposal offers a paradigm shift in aptamer discovery that overcomes many of the roadblocks to aptamer selection. The proposed, genome-inspired approach reverses the selection, using specific DNA sequences from the human genome to capture proteins from natural pools such as nuclear protein extracts. This approach takes advantage of eons of biological evolution that produced genomic DNA sequences that selectively bind to proteins to perform biological functions. Specifically, the goal is to identify proteins that specifically bind to G-quadruplex (G4) sequences in breast cancer gene promoter regions. Although G4 is a highly successful aptamer motif, it is underrepresented in combinatorial libraries; those present are limited to two-tier G4 structures and are prone to inefficient PCR amplification. In contrast, there is a large diversity of multi-tiered G4 structure throughout the human genome including promoter regions of breast cancer genes. The proposed approach mines these previously untapped structures, opening the door to aptamers to new protein targets. Specific Aim 1 is to systematically evaluate the proposed reverse selection approach using genomic G4 forming sequences from the promoter regions of breast cancer-related human genes to capture proteins extracted from cell nuclei of cultured breast cancer and normal breast tissue cell lines. Specific Aim 2 is to perform chromatin immunoprecipitation (ChIP) to determine if a protein selected in vitro binds to the gene promoter regions containing the corresponding G4-forming sequence in the chromatin of live, cultured cells. The research will lead to aptamers to proteins involved in nuclear regulatory processes related to breast cancer, possibly including proteins not yet recognized to be important targets for SELEX.