Genome-Inspired Pathway to Aptamer Discovery

基因组启发的适体发现途径

• 批准号: 8961141
• 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
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8961141
• 关键词: Affect; Affinity; Antibodies; Binding; Biological; Biological Process; Boxing; Breast Cancer gene; 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; In Vitro; Incubated; Investments; Lead; Libraries; Life; Ligands; Mainstreaming; Malignant Neoplasms; Mammary Gland Parenchyma; Mass Spectrum Analysis; Medical; Methods; Mining; Nature; Nuclear; Nuclear Protein; Oligonucleotides; Pathway interactions; Polyacrylamide Gel Electrophoresis; Problem Solving; Process; Promoter Regions; Protein Binding; Proteins; Reagent; Reporting; Research; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Structure; Surveys; Time; Western Blotting; aptamer; chromatin immunoprecipitation; combinatorial; human DNA sequencing; human genome sequencing; magnetic beads; malignant breast neoplasm; next generation sequencing; novel strategies; public health relevance; research study; screening; small molecule; success; tool

 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（由应用提供）：自1990年引入以来，适体已成为抗体越来越多的主流替代品，作为靶标的亲和力试剂，从小分子到大蛋白，甚至整个细胞。它们比抗体具有重要的优势，使它们成为大型分析和医疗应用的有吸引力的候选人。 apatamer发现的过程始于构建“随机”寡核苷酸库，该寡核苷酸库通过迭代选择过程减少，通过指数富集（SELEX）将配体的系统演变（SELEX）降低到对目标高亲和力的少数序列。毫无疑问，Aptamers曾经发现，可以兑现他们的诺言。实现其全部潜力的限制因素是，尽管大量努力和大量时间和金钱投资，但已选择了适体的蛋白质靶标数量。这些失败归因于许多因素，包括SELEX过程本身，众所周知，这些因素是实验室，非标准化且难以完全自动化的。然而，即使解决了这些问题，SELEX仍然存在根本的困难，这将继续阻碍Atamer Discovery的进步。其中包括组合文库的质量，某些结构基序的代表性不足以及PCR扩增的效率低下和偏见。该提案提供了Apattern Discovery的范式转变，该范式将许多障碍的障碍替换为Apattern选择。提出的，基因组启发的方法使用人类基因组的特定DNA序列逆转了选择，从而从天然池（例如核蛋白提取物）捕获蛋白质。这种方法利用了生物进化的Eon，产生了基因组DNA序列，这些基因组DNA序列有选择地结合蛋白质以执行生物学功能。具体来说，目标 尽管G4是一个非常成功的APATLER主题，但在组合库中的代表性不足。存在的人仅限于两层G4结构，并且容易效率低下的PCR扩增。相比之下，整个人类基因组的多层G4结构都有大量多样性，包括乳腺癌基因的启动子区域。提出的方法将这些先前未开发的结构挖掘出来，为新蛋白质靶标的适体打开了大门。具体目的1是系统地评估与乳腺癌相关人基因启动子区域的基因组G4形成序列的拟议反向选择方法，以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是执行染色质免疫沉淀（CHIP），以确定在活体培养细胞的染色质蛋白中，在体外选择的蛋白质是否与含有相应G4形成序列的基因启动子区域结合。这项研究将导致适体与乳腺癌相关的核调节过程，可能包括尚未公认为SELEX的重要靶标的蛋白质。