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

新型锂和钠 DNAzyme 传感器可了解锂治疗双相情感障碍的细胞和分子机制

• 批准号: 9306205
• 负责人: Yi Lu
• 金额: $ 18.31万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9306205
• 关键词: Adverse effects; Affect; Area; Binding; Biochemical; Biology; Biosensor; Bipolar Disorder; Catalytic DNA; Catalytic RNA; Cell model; Cells; Cellular biology; Cleaved cell; Clinical; DNA Library; Depressive disorder; Detection; Disease; Event; Fibroblasts; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Image; In Vitro; Ions; Legal patent; Link; Lithium; Measures; Mental Health; Mental disorders; Metals; Methodology; Methods; Molecular; Monitor; Pathologic; Pathway interactions; Patient-Focused Outcomes; Patients; Physiological; Physiology; Play; Process; Reaction; Reporting; Ribonucleotides; Role; Signal Transduction; Sodium; Specificity; System; Therapeutic; Therapeutic Effect; Transfection; Vertebral column; base; biomaterial compatibility; biophysical techniques; clinical diagnostics; combinatorial; design; divalent metal; dosage; effective therapy; fluorophore; improved; innovation; insight; lymphoblast; metalloenzyme; novel; phosphodiester; ratiometric; response; scaffold; sensor; success; uptake; water solubility; Adverse effects; Affect; Area; Binding; Biochemical; Biology; Biosensor; Bipolar Disorder; Catalytic DNA; Catalytic RNA; Cell model; Cells; Cellular biology; Cleaved cell; Clinical; DNA Library; Depressive disorder; Detection; Disease; Event; Fibroblasts; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Image; In Vitro; Ions; Legal patent; Link; Lithium; Measures; Mental Health; Mental disorders; Metals; Methodology; Methods; Molecular; Monitor; Pathologic; Pathway interactions; Patient-Focused Outcomes; Patients; Physiological; Physiology; Play; Process; Reaction; Reporting; Ribonucleotides; Role; Signal Transduction; Sodium; Specificity; System; Therapeutic; Therapeutic Effect; Transfection; Vertebral column; base; biomaterial compatibility; biophysical techniques; clinical diagnostics; combinatorial; design; divalent metal; dosage; effective therapy; fluorophore; improved; innovation; insight; lymphoblast; metalloenzyme; novel; phosphodiester; ratiometric; response; scaffold; sensor; success; uptake; water solubility

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.

项目摘要/摘要 躁郁症（BD）和其他抑郁症是最常见的精神 影响数百万人的疾病。主要治疗锂（Li+）一直保持 不变> 50年。但是，LI+的治疗窗口非常狭窄（0.5-1.2毫米） 而且副作用可能很严重。同样，Na+失调与BD及其途径有关 也参与了Li+摄取，但对BD或机制的基础知之甚少 Li+的作用。最大化治疗效果并最小化侧面的关键障碍 效应是li+和na+在BD细胞中的浓度和分布的信息缺失， 由于缺乏活细胞中LI+和Na+的选择性传感器。该R21提案试图 探索并展示一类新型DNAZyme传感器的概念证明 Li+或Na+的特异性，用于同时检测BD细胞中LI+和Na+的特异性，以便为了 提供有关BD LI+处理的细胞和分子机制的新见解。 具体而言，我们建议使用一种体外选择方法获得LI+ - 或Na+特异性 dnazymes具有高催化活性的核糖核苷酸裂解。这些序列将是 对传感器应用的特征和优化，因此我们将它们转换为 荧光传感器使用催化信标方法同时成像LI+和Na+ 在BD的淋巴细胞细胞模型中，包括从BD-和非BD患者获得的细胞。这些 荧光传感器将通过掺入既有光值式式传感器进一步改善 在细胞转染期间预防核糖核苷酸裂解的组，一对fret对 荧光团使比率传感能够更好地定量离子浓度。 虽然大多数细胞传感器都设计用于检测二价金属离子（例如 Ca2+），很少开发用于单价离子的有效细胞传感器。通过开发一个 新型的Dnazyme传感器同时提供LI+和Na+的直接度量 BD单元格，该提案提供了一个严重的丢失信息，以提供洞察力 LI+ BD处理的细胞和分子机制。在此过程中，我们将 展示了适用于任何金属离子的基于Dnazyme的传感器的一般方法论 包括使用体外选择结合否定选择以提高选择性和 使用催化信标系统将金属结合转化为不同的荧光 读数。所证明的方法可用于检测和成像LI+和Na+ 对于其他BD单元，可以推广以开发出相似的传感器来成像许多其他金属 离子将推进心理健康，细胞生物学和临床诊断领域。