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

基本信息

批准号：
8705517
负责人：
Laising Lewis Yen
金额：
$ 34.05万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8705517
关键词：
3&apos Untranslated Regions 5&apos 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

项目摘要

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.

描述（由申请人提供）：由于现有技术的限制，尚未完全利用的成像应用的一个重要领域是监测体内特定细胞蛋白的表达的能力。与解剖成像不同，特定细胞蛋白的分子成像会显示出疾病潜在的生化异常，而不是异常的结构后果。如果完全开发，它可以通过对特定标记蛋白的表达进行成像，可以提供监测临床治疗的进展，甚至可能形成一个重要的平台，以实现新的治疗范式的测试和开发。该赠款建议的重点是利用基于RNA的传感器的功能，该传感器将对活细胞中特定的分子特征进行敏感检测。我们设计的RNA传感器通过Polya信号介导的裂解来控制报告基因的表达。哺乳动物Polya信号专门位于3'-非翻译区域（UTR）。当在5'UTR处人为地创建新的Polya位点，在该位点中，它们从不定位在正常的转录单元中时，该Polya信号的极有效裂解会导致mRNA的破坏，从而导致报告基因表达的丧失。靶蛋白与工程polya信号的结合有效地阻断了裂解，从而保留了完整的mRNA，从而实现了记者的表达。反过来，我们已经表明，来自这种传感器的记者信号在活体细胞中表现出极低的泄漏表达，并且在检测特定蛋白质时，该信号被有效地诱导了一百倍以上。这是两个数量级的比以前在活细胞中实现的数量级，具有动态范围，可以在各种实验环境中进行前所未有的应用。总体目的是基于可以利用当前或下一代记者和适体的Polya裂解的调制来创建一个通用分子传感器平台，以成像活细胞中的各种特定分子。此外，已建立的分子传感器平台将为扩展Polya传感器可以在体内检测的分子特征范围提供基础。