Novel, single-molecule aptamer selection method

描述(由申请人提供)： 分子识别是许多生物医学应用的基本原理，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，以及发现靶向特定的候选药物。目前，用于识别研究和诊断配体的主要技术是通过杂交瘤筛选来选择单抗。适配子--一种新兴的抗体模拟物--被使用的程度要小得多，但正受到学术界的关注。适配子是折叠成靶标特异性3D结构的短寡核苷酸。尽管适配子与抗体相比有许多潜在的优势，但它们的应用一直很缓慢，因为1)许多研究人员认为SELEX是选择适配子的唯一方法，2)SELEX被许多人认为是一种繁琐、劳动密集型和耗时的方法，需要专门的专业知识，以及3)SELEX受到严格执行的专利的严格保护。这项提议的长期目标是开发一种方便的、全自动化的基于仪器的系统，用于快速选择在癌症分子分析中有用的适体。NanoMedica计划将一种负担得起的模块化系统商业化，这将使长凳科学家能够选择和表征用于研究癌症、信号转导和蛋白质-蛋白质相互作用的高亲和力适配子。如果成功，这个系统将比SELEX更快、更有效、更易于广泛传播。这项技术的核心是独特的仪器，它结合了纳米操纵器-原子力显微镜(NM-AFM)和倒置光学显微镜，实现了快速成像、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特定结合到固定目标分子的单个适配子。荧光信号将被用来引导AFM尖端朝向结合的适配子，从而实现单分子检测和适配子提取。在提取之前，将通过动态力光谱原位测量适配子与其靶的亲和力。提取后，用聚合酶链式反应扩增适体分子，并用传统的生化方法对其进行进一步鉴定。

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.