Detection strategies:Aptamer-based target recognition to turn-on GOx signaling

检测策略：基于适配体的目标识别开启GOx信号

• 批准号: 8414207
• 负责人: Mehnaaz Fatima Ali
• 金额: $ 14.13万
• 依托单位: XAVIER UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8414207
• 关键词: Affinity; Apoenzymes; Attenuated; Base Pair Mismatch; Binding; Biochemical; Biological Assay; Biological Markers; Biosensor; Calorimetry; Clinical; Complex; Coupled; Data; Detection; Development; Devices; Diagnostic Procedure; Disease; Enzyme Activation; Enzyme Reactivation; Enzymes; Essential Amino Acids; Event; Excision; Faculty; Flavin-Adenine Dinucleotide; Fluorescence Spectroscopy; Generations; Goals; Gold; Health; Home environment; Hydrogen Peroxide; Lead; Link; Malignant Neoplasms; Marriage; Measurable; Measurement; Measures; Mentors; Methods; MicroRNAs; Mission; Natural regeneration; Oligonucleotides; Oncogenic; Outcome; Output; Oxidation-Reduction; Pilot Projects; Production; Property; Reading; Research; Research Personnel; Signal Transduction; Solid; System; Technology; Time; Titrations; Tryptophan; United States National Institutes of Health; Vision; Work; analog; aptamer; base; burden of illness; cofactor; design; enzyme activity; enzyme structure; fluorophore; glucose monitor; glucose oxidase; innovation; member; molecular recognition; novel; point-of-care diagnostics; public health relevance; research study; response; scaffold; sensor; small molecule; tool

DESCRIPTION (provided by applicant): Detection platforms that are adaptable to numerous target types with easy-to-read signal outputs are attractive because of their potential to be used as real-time sensors. A longstanding biosensor and the gold standard for real-time end user devices is the home glucose meter. Central to this device is the enzyme glucose oxidase (GOx), which upon recognition of its substrate results in the production of hydrogen peroxide that is easily measured via electrochemical methods. The redox properties of GOx are dependent on the presence of its co-factor flavin adenine dinucleotide (FAD). The removal of FAD reversibly renders the enzyme inactive without permanently denaturing the enzyme structure. Thus, addition of FAD can be used to 'turn-on' the activity of the enzyme. The long-term goal of this project is to develop novel electrochemical biosensors based on aptamer based target recognition linked with a modulated GOx signaling system. The marriage between these two existing strategies will produce an adaptable detection system that is easily amenable to numerous target types with the real-time signaling capabilities of the GOx signaling system. The objective of this SC2 pilot application is to generate preliminary data showing efficacy of the proposed enzyme reactivated signaling strategy in tandem with aptamer-based molecular recognition. The central hypothesis of this application, which we will pursue via two separate but complementary strategies (Aim 1 and 2 respectively), is that a target- binding event via an aptamer probe will result in the direct release of an enzyme activation trigger. The specific aims for this project are to establish, a detection strategy based on aptamer target binding events for small molecules (Aim 1) and for oligonucleotide target sequences (Aim 2). In both aims, a target-aptamer binding event will release a trigger (either modified or unmodified FAD), which will activate the GOx signaling system. In order to generate preliminary data and prove efficacy of the proposed strategies we will utilize tryptophan (an essential amino acid and important biochemical precursor) as a small-molecule target for Aim 1 and miR-21 (an important oncogenic microRNA implicated in the progression of numerous cancers) as the target sequence for Aim 2. In both aims, we will use enzyme based colorimetric assays (measurable via spectroscopic experiments) to determine the initial rates for the activated enzymes. Subsequently we will measure the binding affinity of the target (i.e. trigger) molecules to their respective aptamers via isothermal titration calorimetry. We will also carry out important control experiments to determine the analytical viability of these detection strategies with respect to selectivity and sensitivity. This proposal is innovative because it develops the first examples of using aptamer-based molecular recognition to directly turn-on the activity of attenuated GOx (apo-GOx). This work is significant because it will provide the preliminary data necessary to show proof-of-concept and will enable us to adapt the current research strategy to an electrochemical solid scaffold at the SC1 level with the long-term vision of developing real-time biosensors for multi-target detection. As importantly it will provide the appropriate mentoring needed to aid the development of the PI to the next level of being an independent researcher and faculty member.

描述（由申请人提供）：具有易于读取的信号输出的适用于多种目标类型的检测平台是有吸引力的，因为它们有可能用作实时传感器。家用血糖仪是一种长期存在的生物传感器，也是实时终端用户设备的黄金标准。该装置的核心是葡萄糖氧化酶（GOx），其在识别其底物后导致过氧化氢的产生，该过氧化氢易于通过电化学方法测量。GOx的氧化还原特性取决于其辅因子黄素腺嘌呤二核苷酸（FAD）的存在。FAD的去除可逆地使酶失活，而不会使酶结构永久变性。因此，添加FAD可用于“开启"酶的活性。该项目的长期目标是开发基于适体的新型电化学生物传感器，该适体与调节的GOx信号系统相关联。这两种现有策略之间的结合将产生一种适应性强的检测系统，该系统易于适用于具有GOx信号系统的实时信号传递能力的多种目标类型。该SC2试点应用的目的是生成初步数据，显示所提出的酶再活化信号传导策略与基于适体的分子识别串联的功效。本申请的中心假设（我们将通过两种单独但互补的策略（分别为目的1和2）来实现）是通过适体探针的靶结合事件将导致酶活化触发剂的直接释放。该项目的具体目标是建立一种基于小分子（目标1）和寡核苷酸靶序列（目标2）的适体靶结合事件的检测策略。在这两个目标中，靶适体结合事件将释放触发物（修饰的或未修饰的FAD），其将激活GOx信号传导系统。为了生成初步数据并证明所提出的策略的功效，我们将利用色氨酸（必需氨基酸和重要的生物化学前体）作为Aim 1的小分子靶标，并利用miR-21（与许多癌症的进展有关的重要致癌microRNA）作为Aim 2的靶序列。在这两个目标中，我们将使用基于酶的比色测定（通过光谱实验可测量）来确定活化酶的初始速率。随后，我们将通过等温滴定量热法测量靶（即触发）分子对其各自适体的结合亲和力。我们还将进行重要的控制实验，以确定这些检测策略在选择性和灵敏度方面的分析可行性。该提议是创新的，因为它开发了使用基于适体的分子识别来直接开启减毒GOx（apo-GOx）活性的第一个实例。这项工作是重要的，因为它将提供必要的初步数据，以显示概念验证，并将使我们能够适应目前的研究策略，在SC 1水平的电化学固体支架与开发实时生物传感器的长期愿景多目标检测。同样重要的是，它将提供适当的指导，以帮助PI的发展成为一个独立的研究人员和教师的下一个水平。