Novel DNAzyme sensors for lithium and sodium to understand cellular and molecular mechanisms of lithium treatment of bipolar disorder

项目摘要/摘要 双相情感障碍(BD)和其他抑郁障碍是最常见的精神疾病 影响数百万人的疾病。主要的治疗，锂(Li)，仍然存在 50年来没有改变。然而，LI的治疗窗口非常窄(0.5-1.2毫米) 而且副作用可能会很严重。此外，钠调节失调与BD及其途径有关 也参与了锂的吸收，但对BD的基础或机制知之甚少 李的行动。使治疗效果最大化和副作用最小化的关键障碍 其作用是丢失了BD细胞中Li和Na的浓度和分布的信息， 因为活细胞中缺乏对Li和Na的选择性传感器。这份R21提案旨在 探索和演示一类新型高性能脱氧核酶传感器的概念验证 Li或Na在BD细胞中同时检测Li和Na的特异性 为LI治疗BD的细胞和分子机制提供新的见解。 具体地说，我们建议使用体外选择方法来获得Li或Na特异的 对核糖核酸具有高催化活性的脱氧核酶。这些序列将是 针对传感器应用进行了特征化和优化，因此我们将把它们转化为 催化信标法同时成像锂和钠的荧光传感器 在BD的淋巴母细胞模型中，包括从BD患者和非BD患者获得的细胞。这些 荧光传感器将进一步改进，通过结合光敏笼式 保护细胞在细胞转染过程中不被核苷酸切割的组，以及一对FRET 荧光团使比率传感成为可能，以便更好地量化离子浓度。 虽然大多数蜂窝传感器都是为检测二价金属离子而设计的(例如， Ca2)，目前对单价离子的有效细胞传感器还很少。通过开发一种 一种新型的DNAzyme传感器，可同时提供Li和Na的直接测量 BD细胞，这项建议提供了一个关键的缺失信息，以提供对 LI治疗BD的细胞和分子机制。在这个过程中，我们将 演示基于DNAzyme的传感器适用于任何金属离子的一般方法， 包括使用体外选择结合负选择来提高选择性和 利用催化信标系统将金属结合转化为不同的荧光 读数。该方法可用于锂、钠的检测和成像 用于其他BD细胞，并可推广到开发类似的传感器以成像许多其他金属 离子，这将推动心理健康、细胞生物学和临床诊断领域的发展。

Project Summary/Abstract Bipolar disorder (BD) and other depressive disorders are some of the most common mental illnesses affecting millions of people. The primary treatment, lithium (Li+), has remained unchanged for > 50 years. However, the therapeutic window for Li+ is very narrow (0.5-1.2 mM) and the side effects can be severe. Also, Na+ dysregulation is implicated in BD and its pathways are also involved in Li+ uptake, but very little is known about the basis of BD or the mechanism of action of Li+. A critical barrier to maximizing the therapeutic effect and minimizing the side effect is missing information of the concentrations and distributions of Li+ and Na+ in BD cells, because of lack of selective sensors for Li+ and Na+ in living cells. This R21 proposal seeks to explore and demonstrate proof-of-concept of a novel class of DNAzyme sensors with high specificity for either Li+ or Na+ for simultaneous detection of Li+ and Na+ in BD cells, in order to provide new insights into the cellular and molecular mechanisms of Li+ treatment of BD. Specifically, we propose to use an in vitro selection method to obtain Li+- or Na+-specific DNAzymes with high catalytic activity towards ribonucleotide cleavage. These sequences will be characterized and optimized for sensor applications, whereupon we will transform them into fluorescent sensors using the catalytic beacon method for simultaneous imaging of Li+ and Na+ in lymphoblast cell models of BD, including cells obtained from BD- and non-BD patients. These fluorescent sensors will further be improved upon by incorporating both a photolabile-caging group to protect against ribonucleotide cleavage during the cell transfection, and a FRET pair of fluorophores to enable ratiometric sensing to allow for better quantitation of ion concentrations. While most cellular sensors have been designed for detection of divalent metal ions (e.g. Ca2+), few effective cellular sensors have been developed for monovalent ions. By developing a novel class of DNAzyme sensors to provide a direct measure of Li+ and Na+ simultaneously in BD cells, this proposal provides a critical missing piece of information in providing insights into the cellular and molecular mechanisms for Li+ treatment of BD. In the process, we will demonstrate general methodologies for DNAzyme-based sensors applicable for any metal ion, including the use of in vitro selection incorporating negative selection to improve selectivity and use of the catalytic beacon system to transform metal binding into different fluorescence readouts. The methods demonstrated can be applied for detection and imaging of Li+ and Na+ for other BD cells and can be generalized to develop similar sensors to image many other metal ions, which will advance the fields of mental health, cell biology, and clinical diagnostics.