Modified RNA tools and diagnostics for drug abuse

描述(申请人提供)：项目摘要/摘要RNA修饰很多，范围从简单的甲基化到复杂的变化，如添加氨基酰氨基甲酰基。这些修饰及其存在的水平正在成为生物过程的重要调节因素，而化学或药物暴露等扰动可以改变RNA亚型上的特定修饰模式。此外，考虑到它们的多样性、特异性及其与功能和结构稳定性的关系，RNA修饰是疾病和治疗进展的潜在有价值的生物标记物，特别是考虑到修饰的核苷即使在RNA降解后也相对稳定，并可以从细胞释放到体液中。这项研究的长期目标是促进对RNA修饰及其对健康和疾病影响的研究。直接的障碍和这项应用的目标是开发极其灵敏的分析方法来检测和量化常见的和新的RNA修饰，这些修饰可以很容易地传播给RNA研究界。这个测试案例的生物系统是基于人类诱导的多能干细胞来源的神经系统细胞，这些细胞将暴露在两种不同的滥用药物尼古丁和大麻类药物中。目的1.我们将使用RNA修饰的核苷标准品来开发高灵敏度和强健的分析方法。我们将开发的最终工具是一种产品，它是两种技术--UHPLC和MS--相结合的产物，将针对高分辨率分离和高灵敏度的RNA修饰检测进行优化。所开发的工具将在分化的大脑皮层神经元和神经胶质细胞上进行测试，并将对不同成熟阶段的RNA修饰进行表征和丰度量化。目的2.在AIM 1期间开发的工具将进一步适用于检测特定的RNA修饰，这些修饰可能因滥用药物治疗而对分化的前脑细胞发挥重要作用。在这个目标中，我们将专门研究暴露在不同尼古丁和大麻类药物浓度下的前脑细胞，并与车辆对照组进行比较。我们提议的方法的结果将以高度灵敏和准确的工具的形式用于分析调控RNA中存在的广泛的化学成分和大量的RNA修饰。最终目标是开发高度敏感的基于RNA修饰的生物标记物发现工具，这种工具需要最低水平的技术专业知识，作为传播和加速RNA修饰领域的一种方式，以获得对疾病机制的进一步了解。

DESCRIPTION (provided by applicant): Project Summary/Abstract RNA modifications are numerous, and range from simple methylation to complex changes such as addition of aminoacylcarbamoyl moieties. These modifications and, the levels at which are present, are emerging as important regulators of biological processes, and perturbations such as chemical or drug exposure can alter the patterns of specific modifications on RNA sub-types. Moreover, given their variety, specificity and their relationship to function and structure stability, RNA modifications are potentially valuable biomarkers of disease and treatment progression, especially given that modified nucleosides are relatively stable even after RNA degradation, and can be released from cells into bodily fluids. The long term objective of this study is to facilitae research into RNA modifications and their impact on health and disease. The immediate hurdle, and the goal of this application, is to develop extremely sensitive analytical methods for detection and quantification of common and novel RNA modifications that can be readily disseminated to the RNA research community. The test case biological system is based on human induced pluripotent stem cell derived nervous system cells that will be exposed to two different drugs of abuse, nicotine and cannabinoids. Aim 1. We will use RNA modified nucleoside standards to develop highly sensitive and robust method of analysis. The ultimate tool we will be developing is a product resultant from the combination of two technologies, UHPLC and MS that will be optimized for high resolution separation and highly sensitive detection of RNA modifications. The tools developed will be tested on differentiated anterior cerebral cortex neurons and glia cells and RNA modifications will be characterized and abundances quantified during the different stages of maturation. Aim 2. Tools developed during aim 1 will be further adapted to detect specific RNA modifications that may be playing a significant role on differentiated forebrain cells, as a result from treatment with drug of abuse. During this aim we will specifically study forebrain cells that will be exposed to different nicotie and cannabinoids concentrations and compared to vehicle controls. The results of the methods we are proposing will be in the form of highly sensitive and accurate tools for the analysis of a wide range of chemistries and abundances of RNA modifications present in regulatory RNAs. The ultimately goal is to develop highly sensitive RNA modifications-based biomarkers discovery tools that requires minimal level of technical expertise as a way to disseminate and accelerate the field of RNA modifications with regards to gain further insight into mechanisms of disease.