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

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.

项目总结/摘要 双相情感障碍（BD）和其他抑郁症是一些最常见的精神疾病， 影响数百万人的疾病。主要的治疗方法，锂（Li+）， 50年不变。然而，Li+的治疗窗口非常窄（0.5-1.2 mM） 副作用可能很严重此外，Na+失调与BD及其途径有关 也参与了Li+的吸收，但对BD的基础或机制知之甚少 Li+的作用。最大化治疗效果和最小化副作用的关键障碍 影响是缺少BD细胞中Li+和Na+的浓度和分布的信息， 因为活细胞中缺乏Li+和Na+的选择性传感器。R21提案旨在 探索并展示了一类新型DNAzyme传感器的概念验证， 同时检测BD细胞中Li+和Na+的Li+或Na+特异性， 为Li+治疗BD的细胞和分子机制提供了新的见解。 具体而言，我们建议使用体外选择方法来获得Li+或Na+特异性的 对核糖核苷酸切割具有高催化活性的DNA酶。这些序列将是 针对传感器应用进行了特性化和优化，因此我们将把它们转换为 用于Li+和Na+同时成像的使用催化信标方法的荧光传感器 在BD的淋巴母细胞模型中，包括从BD和非BD患者获得的细胞。这些 荧光传感器将通过结合光敏感性-笼化 在细胞转染过程中保护核糖核苷酸不被切割的FRET对， 荧光团，以实现比率传感，从而允许更好地定量离子浓度。 虽然大多数细胞传感器已被设计用于检测二价金属离子（例如， Ca 2+），但很少有有效的细胞传感器已开发的单价离子。通过开发一个 一种新型的DNAzyme传感器，可同时直接测量Li+和Na+， BD细胞，这项建议提供了一个关键的缺失信息，在提供见解， Li+治疗BD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DNA酶的传感器的一般方法， 包括使用结合负选择的体外选择以提高选择性， 使用催化信标系统将金属结合转化为不同的荧光 读数所展示的方法可应用于Li+和Na+的检测和成像 并且可以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。

项目成果

Evidence of a General Acid-Base Catalysis Mechanism in the 8-17 DNAzyme.

• DOI: 10.1021/acs.biochem.7b01096
• 发表时间: 2018-03-06
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Cepeda-Plaza M;McGhee CE;Lu Y
• 通讯作者: Lu Y

Discovery of and Insights into DNA "Codes" for Tunable Morphologies of Metal Nanoparticles.

• DOI: 10.1002/smll.201900975
• 发表时间: 2019-05
• 期刊: Small
• 影响因子: 13.3
• 作者: Nitya Sai Reddy Satyavolu;K. Y. Loh;L. H. Tan;Yi Lu

DNAzyme sensors for detection of metal ions in the environment and imaging them in living cells.

• DOI: 10.1016/j.copbio.2017.03.002
• 发表时间: 2017-06
• 期刊: Current opinion in biotechnology
• 影响因子: 7.7
• 作者: McGhee CE;Loh KY;Lu Y
• 通讯作者: Lu Y

Advancing Point-of-Care Diagnostics of Metabolites Through Engineering Semisynthetic Proteins.

通过工程半合成蛋白质推进代谢物的即时诊断。

• DOI: 10.1373/clinchem.2018.298836
• 发表时间: 2019
• 期刊: Clinical chemistry
• 影响因子: 9.3
• 作者: Zhang,JingJing;Lu,Yi
• 通讯作者: Lu,Yi

Optical Control of Metal Ion Probes in Cells and Zebrafish Using Highly Selective DNAzymes Conjugated to Upconversion Nanoparticles.

• DOI: 10.1021/jacs.8b09867
• 发表时间: 2018-11
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Zhenglin Yang;K. Y. Loh;Yu Chu;Ruopei Feng;Nitya Sai Reddy Satyavolu;Mengyi Xiong;Stephanie M Nakamata Huynh-Stephan