In-vivo miRNA Detection Using Structurally Interacting RNA (sxRNA)

使用结构相互作用 RNA (sxRNA) 进行体内 miRNA 检测

• 批准号: 8199709
• 负责人: SCOTT A TENENBAUM
• 金额: $ 29.36万
• 依托单位: HOCUSLOCUS, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8199709
• 关键词: Affinity; Base Pairing; Binding; Binding Proteins; Biological Assay; Breeding; Cell Death; Cells; Cessation of life; Code; Complex; Custom; Cystic Fibrosis; Deficiency Diseases; Detection; Diagnostic; Disease; Ensure; Functional RNA; Future; Gene Expression; Gene Expression Regulation; Generations; Genes; Goals; Histocompatibility Testing; Histones; Immunoprecipitation; In Vitro; K-562; K562 Cells; Life; Malignant Neoplasms; Measures; Messenger RNA; Methods; MicroRNAs; Molecular; Mutate; Nucleotides; Pattern; Phase; Population; Positioning Attribute; Procedures; Production; Protein Binding; Protein Deficiency; Proteins; RNA; RNA-Binding Proteins; Regulation; Reporter; Reporter Genes; Research Personnel; Scientist; Sensitivity and Specificity; Signal Transduction; Solutions; Specificity; Stem cells; Structure; System; Systems Development; Technology; Testing; Therapeutic; Time; Translations; Undifferentiated; Virus Diseases; Work; base; cancer therapy; cell type; design; gene therapy; in vivo; prevent; protein B; stem; stem cell differentiation; success; tool

DESCRIPTION (provided by applicant): We propose developing a trans-molecular RNA-switch for scientists to measure miRNA activity in-vivo. While there are numerous methods to quantify miRNAs in vitro, we are not aware of any method that allows for in vivo analysis. The ability to observe specific miRNA generation in living single cells without requiring the destruction of the cell would be a significant tool. We are pioneering an RNA-based switch technology called structurally interacting RNAs (sxRNA) which utilizes post-transcriptional gene regulation as a reporter for miRNA detection. Normally, RNA-binding proteins (RBP) associate with a 3' stem-loop structure to facilitate translation of an upstream coding region by as much as an order of magnitude. It is possible to modify the mRNA to modulate translation of the reporter gene by controlling the binding of RBP. This is accomplished by altering the natural stem-loop structure so that it does not form unless an additional RNA, such as a miRNA, binds and stabilizes the functional structure by base- pairing with the flanking regions of the custom designed stem-loop. Our goal with this proposal is first to evaluate the switch mechanism in-vitro and measure the specificity of interaction and signal strength possible. Secondly, we will measure the effect on translation and determine whether this switching mechanism is effective in a cell. Success at the product level using sxRNA as a diagnostic tool should ideally position the technology for additional uses. It could be effective anywhere that miRNA expression levels are an indicator of disease, cell or tissue type, or condition. sxRNA could be an RNA-based alternative to gene therapy for protein deficiency diseases, such as cystic fibrosis, or a cancer therapy. PUBLIC HEALTH RELEVANCE: While there are numerous methods to quantify miRNAs in vitro, we are not aware of any method that allows for in vivo analysis. The ability to observe specific miRNA generation in living single cells without requiring the destruction of the cell would be a significant tool. For example, stem cell researchers could ensure stem cells had not differentiated by in-vivo detection of miRNAs that are preferentially expressed during differentiation.

描述（由申请人提供）：我们建议开发一种跨分子RNA开关，供科学家在体内测量miRNA活性。虽然有许多体外定量miRNA的方法，但我们不知道任何允许体内分析的方法。在活的单细胞中观察特异性miRNA产生而不需要破坏细胞的能力将是一个重要的工具。我们正在开创一种基于RNA的开关技术，称为结构相互作用RNA（sxRNA），它利用转录后基因调控作为miRNA检测的报告基因。通常，RNA结合蛋白（RBP）与3'茎环结构结合，以促进上游编码区的翻译多达一个数量级。有可能通过控制RBP的结合来修饰mRNA以调节报告基因的翻译。这通过改变天然茎环结构来实现，使得除非另外的RNA（例如miRNA）通过与定制设计的茎环的侧翼区碱基配对来结合并稳定功能结构，否则它不会形成。我们的目标是首先在体外评估开关机制，并测量相互作用的特异性和可能的信号强度。其次，我们将测量对翻译的影响，并确定这种转换机制在细胞中是否有效。使用sxRNA作为诊断工具在产品层面的成功应该使该技术在其他用途上处于理想地位。它可以在任何地方有效，miRNA表达水平是疾病，细胞或组织类型或状况的指标。sxRNA可能是一种基于RNA的替代基因治疗蛋白质缺乏性疾病，如囊性纤维化，或癌症治疗。 公共卫生相关性：虽然有许多体外定量miRNA的方法，但我们不知道任何允许体内分析的方法。在活的单细胞中观察特异性miRNA产生而不需要破坏细胞的能力将是一个重要的工具。例如，干细胞研究人员可以通过体内检测在分化过程中优先表达的miRNA来确保干细胞没有分化。