Novel, single-molecule aptamer selection method

新颖的单分子适体选择方法

• 批准号: 6687228
• 负责人: MARTIN GUTHOLD
• 金额: $ 11.37万
• 依托单位: NANOMEDICA, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-06 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6687228
• 关键词: analytical method; atomic force microscopy; fluorescence microscopy; image enhancement; light microscopy; molecular shape; nanotechnology; nucleic acid quantitation /detection; oligonucleotides; technology /technique development

DESCRIPTION (provided by applicant): Molecular recognition is the underlying principle in many biomedical applications, such as labeling and imaging specific proteins, organelles, cells, tissues and organs, detecting and quantifying clinical analytes, and discovering target-specific drug candidates. Currently, the primary technology for identifying research and diagnostic ligands is monoclonal antibody selection by hybridoma screening. Aptamers - an emerging class of antibody mimetics - have been used to a much lesser extent but are gaining attention in academic circles. Aptamers are short oligonucleotides that fold into target-specific 3D structures. Although aptamers have many potential advantages over antibodies, their adoption has been slow, because 1) many researchers presume that SELEX is the only way to select aptamers, 2) SELEX is regarded by many as a cumbersome, labor-intensive and time-consuming method that requires specialized expertise, and 3) SELEX is heavily protected by patents that are aggressively enforced. The long-term goal of this proposal is to develop a convenient and fully automated instrument-based system for rapid selection of aptamers useful in the molecular analysis of cancer. NanoMedica plans to commercialize an affordable, modular system that will empower bench scientists to select and characterize high-affinity aptamers for studying cancer, signal transduction, and protein-protein interactions. If successful, this system will be fast, efficient and much more amenable to broad dissemination than SELEX. At the core of this technique is unique instrumentation that combines a nanoManipulator-Atomic Force Microscope (nM-AFM) with an inverted optical microscope to achieve fast imaging, high resolution and single-molecule detection and manipulation capabilities. The instrument will be able to detect single aptamers specifically bound to immobilized target molecules by fluorescence microscopy. The fluorescence signal will be used to guide the AFM tip towards the bound aptamer to enable single-molecule detection and aptamer extraction. Before extraction, the aptamer's affinity for its target will be measured in situ by dynamic force spectroscopy. After extraction, the aptamer molecule will be amplified by PCR and further characterized by traditional biochemical methods.

描述（由申请人提供）： 分子识别是许多生物医学应用中的基本原理，例如标记和成像特定的蛋白质、细胞器、细胞、组织和器官，检测和定量临床分析物，以及发现靶特异性候选药物。目前，用于鉴定研究和诊断配体的主要技术是通过杂交瘤筛选的单克隆抗体选择。适体--一种新兴的抗体模拟物--的使用程度要小得多，但在学术界越来越受到关注。适体是折叠成靶特异性3D结构的短寡核苷酸。尽管适体相对于抗体具有许多潜在优势，但它们的采用一直很缓慢，因为1）许多研究人员认为SELEX是选择适体的唯一方法，2）许多人认为SELEX是一种繁琐，劳动密集型和耗时的方法，需要专业知识，3）SELEX受到严格执行的专利的严格保护。该提案的长期目标是开发一种方便且完全自动化的基于仪器的系统，用于快速选择适用于癌症分子分析的适体。NanoMedica计划将一个负担得起的模块化系统商业化，该系统将使实验室科学家能够选择和表征高亲和力适体，用于研究癌症，信号转导和蛋白质-蛋白质相互作用。如果成功，这一系统将是快速、有效的，而且比SELEX更适合广泛传播。 这项技术的核心是独特的仪器，它将纳米操纵器-原子力显微镜（nM-AFM）与倒置光学显微镜相结合，以实现快速成像，高分辨率和单分子检测和操纵能力。该仪器将能够通过荧光显微镜检测与固定化靶分子特异性结合的单个适体。荧光信号将用于引导AFM针尖朝向结合的适体，以实现单分子检测和适体提取。在提取之前，将通过动态力光谱法原位测量适体对其靶标的亲和力。提取后，将通过PCR扩增适体分子，并通过传统的生物化学方法进一步表征。