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Genome-Inspired Pathway to Aptamer Discovery

基因组启发的适体发现途径

基本信息

批准号：
9313901
负责人：
Linda B. Mc GOWN
金额：
$ 23.52万
依托单位：
RENSSELAER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9313901
关键词：
Affect Affinity Antibodies Binding Binding Proteins Biological Biological Process Breast Cancer gene Cell Extracts Cell Line Cell Nucleus Cells Chromatin Complement Computing Methodologies Cultured Cells DNA DNA Sequence Development Diagnosis Disease Evolution Exhibits Failure G-Quartets Gene Expression Regulation Genes Genome Genomic DNA Genomics Goals Human Human Genome Immobilization In Vitro Incubated Investments Libraries Ligands Mainstreaming Malignant Neoplasms Mammary Gland Parenchyma Mass Spectrum Analysis Medical Methods Nature Nuclear Nuclear Protein Oligonucleotides Pathway interactions Polyacrylamide Gel Electrophoresis Problem Solving Process Promoter Regions Proteins Reagent Reporting Research Specificity Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Speed Structure Surveys Time Western Blotting aptamer chromatin immunoprecipitation combinatorial experimental study magnetic beads malignant breast neoplasm next generation sequencing novel strategies public health relevance screening small molecule success tool

项目摘要

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.

描述（由申请人提供）：自1990年引入以来，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全细胞等靶点的亲和试剂。与抗体相比，它们具有重要的优势，使其成为广泛的分析和医学应用的有吸引力的候选者。适体发现的过程开始于构建“随机”寡核苷酸文库，该文库通过迭代选择过程（指数富集配体系统进化（SELEX））减少到少数对靶表现出高亲和力的序列。毫无疑问，适体一旦被发现，就可以实现它们的承诺。实现其全部潜力的限制因素是尽管进行了大量的努力和大量的时间和金钱的投资，但已经选择了适体的蛋白质靶标的相对萨尔数量。这些失败归因于许多因素，包括SELEX过程本身，这是出了名的费力，非标准化和难以完全自动化。然而，即使这些问题得到解决，SELEX中仍然存在固有的基本困难，这将继续阻碍适体发现的进展。这些问题包括组合文库的质量、某些结构基序的代表性不足以及PCR扩增的效率和偏差。该提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。这项由基因组启发的方法逆转了这种选择，使用人类基因组中的特定DNA序列从天然库（如核蛋白提取物）中捕获蛋白质。这种方法利用了生物进化过程中产生的基因组DNA序列，这些序列选择性地与蛋白质结合以执行生物功能。具体来说，目标目的是鉴定与乳腺癌基因启动子区域中的G-四链体（G4）序列特异性结合的蛋白质。虽然G4是一个非常成功的适体基序，但它在组合文库中的代表性不足;存在的那些仅限于双层G4结构，并且易于进行低效的PCR扩增。相比之下，在整个人类基因组中存在大量多样性的多层G4结构，包括乳腺癌基因的启动子区域。所提出的方法挖掘了这些以前未开发的结构，为新蛋白质靶点的适体打开了大门。具体目的1是系统地评估所提出的反向选择方法，该方法使用来自乳腺癌相关人类基因的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。具体目标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染色质中含有相应G4形成序列的基因启动子区结合。这项研究将导致与乳腺癌相关的核调节过程中涉及的蛋白质的适体，可能包括尚未被认为是SELEX重要靶点的蛋白质。