Harnessing the power of RNA sensor for imaging molecular signatures in vivo

利用 RNA 传感器的力量对体内分子特征进行成像

• 批准号: 8146820
• 负责人: Laising Lewis Yen
• 金额: $ 36.47万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8146820
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Address; Adenoviruses; Applications Grants; Aptamer Technology; Area; Binding; Biochemical; Biological Preservation; Cell Culture Techniques; Cell Line; Cells; Clinical Treatment; Coupling; Detection; Development; Disease; Elements; Engineering; Ensure; Environment; Exhibits; Foundations; Gene Expression; Gene Transfer; Generic Drugs; Goals; HIV; HIV tat Protein; Human; Image; Label; Life; Ligands; Link; Liver; Mammalian Cell; Measurement; Mediating; Messenger RNA; Methods; Molecular; Molecular Profiling; Monitor; Mus; NF-kappa B; Noise; Non-Invasive Cancer Detection; Polyadenylation; Process; Protein Isoforms; Proteins; RNA; Reporter; Reporter Genes; Reporting; Signal Transduction; Signaling Protein; Site; Staining method; Stains; Structure; System; TNFRSF5 gene; Technology; Testing; Work; aptamer; base; cell type; design; flexibility; in vivo; molecular imaging; next generation; novel therapeutics; portability; sensor; whole body imaging

DESCRIPTION (provided by applicant): One important area of application of imaging that has not been fully exploited, due to limitation of existing technology, is the ability to monitor the expression of specific cellular proteins in vivo. Unlike anatomical imaging, molecular imaging of specific cellular proteins would display the biochemical abnormalities underlying disease rather than the structural consequences of abnormalities. If fully developed, it could offer the opportunity to monitor the progress of clinical treatments by imaging the expression of specific marker proteins, and may even form an important platform to enable the testing and development of new therapeutic paradigms. The focus of this grant proposal is to harness the power of RNA-based sensors that will enable sensitive detection of specific molecular signatures in living cells. The RNA sensor we engineered controls the expression of a reporter gene by polyA signal-mediated cleavage. Mammalian polyA signals are exclusively located at the 3'-untranslated region (UTR). When a new polyA site is artificially created at 5' UTR, where they are never localized in normal transcriptional units, extremely efficient cleavage of that polyA signal leads to destruction of the mRNA and therefore loss of reporter gene expression. Binding of a target protein to the engineered polyA signal efficiently blocks the cleavage, resulting in preservation of the intact mRNA, thus enabling reporter expression. In turn, we have shown that the reporter signal from such a sensor exhibited extremely low leaky expression in live human cells, and upon the detection of a specific protein, the signal was effectively induced above one hundred folds. This is two orders of magnitude higher than has been previously achieved in live human cells, giving a dynamic range that would allow unprecedented applications in a variety of experimental settings. The overall objective is to create a general molecular sensor platform based on the modulation of polyA cleavage that could utilize current or next generation reporters and aptamers for the purpose of imaging a variety of specific molecules in live cells. Moreover, the established molecular sensor platform will provide a foundation for expanding the spectrum of molecular signatures that the polyA sensor can detect in vivo. PUBLIC HEALTH RELEVANCE: This application aims to harness the power of RNA-based sensors that will enable sensitive detection of specific molecular signatures in living cells. The RNA sensor described offers enhanced capacities for imaging molecular signatures in vivo than is currently possible. The application directly answers the needs outlined in PAR-09-016.

描述(申请人提供)：由于现有技术的限制，成像应用的一个重要领域尚未得到充分开发，那就是监测体内特定细胞蛋白的表达的能力。与解剖成像不同，特定细胞蛋白质的分子成像将显示疾病背后的生化异常，而不是异常的结构后果。如果完全开发，它可以提供通过成像特定标记蛋白的表达来监测临床治疗进展的机会，甚至可能形成一个重要的平台，使新的治疗范例的测试和开发成为可能。这项拨款提案的重点是利用基于RNA的传感器的力量，这将使在活细胞中敏感地检测特定的分子特征成为可能。我们设计的RNA传感器通过PolyA信号介导的切割来控制报告基因的表达。哺乳动物的Polya信号仅位于3‘非翻译区(UTR)。当在5‘非编码区人工创建一个新的PolyA位点时，它们永远不会定位在正常的转录单位中，该PolyA信号的极其有效的切割会导致mRNA的破坏，从而失去报告基因的表达。靶蛋白与工程PolyA信号的结合有效地阻止了切割，导致了完整的mRNA的保存，从而使报告基因得以表达。反过来，我们发现来自这种传感器的报告信号在活的人类细胞中表现出极低的泄漏表达，并且在检测到特定蛋白质后，该信号被有效地诱导了100倍以上。这比之前在活体人体细胞中达到的水平高出两个数量级，提供了一个动态范围，将允许在各种实验环境中进行前所未有的应用。总体目标是创建一个基于Polya裂解调制的通用分子传感器平台，该平台可以利用当前或下一代报告和适配子来成像活细胞中的各种特定分子。此外，建立的分子传感器平台将为扩大Polya传感器可以在体内检测到的分子特征谱提供基础。 与公共卫生相关：这项应用旨在利用基于RNA的传感器的力量，使在活细胞中能够灵敏地检测特定的分子特征。所描述的RNA传感器提供了比目前可能的更强的在体内成像分子签名的能力。该应用程序直接满足PAR-09-016中概述的需求。