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

描述(由申请人提供)：可适应多种目标类型、信号输出易于读取的检测平台具有吸引力，因为它们具有用作实时传感器的潜力。家用血糖仪是一种历史悠久的生物传感器，也是实时终端用户设备的黄金标准。这个装置的核心是葡萄糖氧化酶(GOX)，一旦识别其底物，它就会产生过氧化氢，很容易通过电化学方法测量。Gox的氧化还原特性依赖于其辅助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去活性，而不会永久变性酶的结构。因此，添加FAD可以用来“启动”酶的活性。该项目的长期目标是开发基于适体的目标识别与调制的Gox信号系统相连的新型电化学生物传感器。这两种现有战略的结合将产生一种适应性强的检测系统，它很容易适应多种目标类型，具有Gox信令系统的实时信令能力。这项SC2中试应用的目标是产生初步数据，显示所建议的酶重新激活信号策略与基于适体的分子识别相结合的有效性。这项应用的中心假设，我们将通过两个独立但互补的策略(分别为目标1和目标2)进行，即通过适体探针的靶结合事件将导致酶激活触发的直接释放。本项目的具体目标是建立一种基于小分子适体靶标结合事件(目标1)和寡核苷酸靶标序列(目标2)的检测策略。在这两个AIM中，靶-适体结合事件将释放一个触发器(修改或未修改的FAD)，从而激活Gox信号系统。为了生成初步数据并证明建议策略的有效性，我们将使用色氨酸(一种必需的氨基酸和重要的生化前体)作为Aim 1的小分子靶标，将miR-21(一种重要的致癌微RNA，参与许多癌症的进展)作为Aim 2的目标序列。在这两个Aim中，我们将使用基于酶的比色分析(可通过光谱实验测量)来确定激活酶的初始速率。随后，我们将通过等温滴定量热法测量目标(即触发)分子与其各自适配子的结合亲和力。我们还将进行重要的对照实验，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。这项提议是创新的，因为它开发了第一个使用基于适体的分子识别来直接启动衰减的Gox(apo-Gox)活性的例子。这项工作意义重大，因为它将提供展示概念验证所需的初步数据，并使我们能够将当前的研究策略调整到SC1水平的电化学固体支架上，并着眼于开发用于多目标检测的实时生物传感器。同样重要的是，它将提供所需的适当指导，以帮助PI发展到成为独立研究人员和教员的下一个水平。

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.