Chemical Probes of Gene Regulatory Mechanisms

基因调控机制的化学探针

• 批准号: 7734290
• 负责人: Hans Luecke
• 金额: $ 15.68万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734290
• 关键词: Active Sites; Cells; Chemicals; Chemistry; Code; Condition; Copper; Cultured Cells; Dissection; Fluorescence Microscopy; Functional RNA; Gene Expression Regulation; Genetic Transcription; Genome; Genomics; Goals; Growth; Label; Life; Measures; Messenger RNA; Methods; Nucleosides; Oxidative Stress; Pattern; Polymerase Chain Reaction; Process; RNA; RNA chemical synthesis; Rate; Regulation; Regulator Genes; Relative (related person); Ribonucleosides; Stimulus; Synthesis Chemistry; analog; base; cDNA Arrays; cytokine; fluorescence imaging; human tissue; interdisciplinary approach; interest; small molecule; tissue culture

Our approach involves labeling cellular RNA using chemically modified ribonucleoside analogs. We have developed a method to introduce these modified bases into RNA of living cells following short treatment with the modified nucleosides. The labels introduced into RNA are chemically derivatized using copper catalyzed click chemistry, which allows us to distinguish and physically separate newly synthesized RNA molecules (coding and non-coding) from mature RNA. We are using this approach to visualize sites of active transcription in living cells using fluorescence microscopy, and to determine the turnover rates of RNA molecules across the genome using quantitative PCR, microarray and cDNA sequencing. These approaches allow us to compare genomic regulatory patterns under a variety of stimuli and growth conditions, including, cytokine stimulation, oxidative stress and treatment with pharmacologically active small molecules.

我们的方法涉及使用化学修饰的核糖核苷类似物标记细胞RNA。 我们已经开发了一种方法，将这些修饰的碱基引入活细胞的RNA中，然后用修饰的核苷进行短时间处理。 引入RNA的标记使用铜催化的点击化学进行化学衍生，这使我们能够区分和物理分离新合成的RNA分子（编码和非编码）与成熟RNA。 我们正在使用这种方法来可视化网站的活性转录活细胞使用荧光显微镜，并确定周转率的RNA分子在整个基因组使用定量PCR，微阵列和cDNA测序。 这些方法使我们能够比较各种刺激和生长条件下的基因组调控模式，包括细胞因子刺激，氧化应激和治疗与抗肿瘤活性小分子。