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

项目概要/摘要 双相情感障碍 (BD) 和其他抑郁症是最常见的精神障碍之一 影响数百万人的疾病。主要治疗方法，锂（Li），仍然保留 超过 50 年没有变化。然而，Li的治疗窗口非常窄（0.5-1.2 mM） 并且副作用可能很严重。此外，Na 失调也与 BD 及其通路有关 也参与锂的吸收，但对 BD 的基础或机制知之甚少 李的行动。最大化治疗效果和最小化副作用的关键障碍 效应是缺少 BD 细胞中 Li 和 Na 的浓度和分布信息， 因为活细胞中缺乏对锂和钠的选择性传感器。该 R21 提案旨在 探索并展示一类新型 DNAzyme 传感器的概念验证，具有高 对 Li 或 Na 的特异性，可同时检测 BD 细胞中的 Li 和 Na，以便 为Li治疗BD的细胞和分子机制提供新的见解。 具体来说，我们建议使用体外选择方法来获得 Li 或 Na 特异性 对核糖核苷酸裂解具有高催化活性的脱氧核糖核酸酶。这些序列将是 针对传感器应用进行特征化和优化，因此我们将它们转化为 使用催化信标法同时成像锂和钠的荧光传感器 BD 的淋巴细胞模型，包括从 BD 和非 BD 患者获得的细胞。这些 通过结合光不稳定笼，荧光传感器将得到进一步改进 组以防止细胞转染期间核糖核苷酸裂解，以及一对 FRET 荧光团可实现比率传感，从而更好地定量离子浓度。 虽然大多数细胞传感器被设计用于检测二价金属离子（例如， Ca2 ），很少有人开发出针对单价离子的有效细胞传感器。通过开发一个 新型 DNAzyme 传感器可同时直接测量 Li 和 Na BD 细胞，该提案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。在此过程中，我们将 展示适用于任何金属离子的基于 DNAzyme 的传感器的通用方法， 包括使用体外选择结合负选择来提高选择性和 使用催化信标系统将金属结合转化为不同的荧光 读数。所演示的方法可用于锂和钠的检测和成像 适用于其他 BD 电池，并且可以推广到开发类似的传感器来对许多其他金属进行成像 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.