Engineered circular RNAs: A novel platform for genetically encoded RNA-based biosensors and their application as real-time viral infection reporters

工程环状RNA：基于基因编码RNA的生物传感器的新型平台及其作为实时病毒感染报告基因的应用

• 批准号: 9397184
• 负责人: Jacob Litke
• 金额: $ 4.4万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-31 至 2020-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9397184
• 关键词: Area; Behavior; Binding; Biosensor; Biotechnology; Catalytic RNA; Cell Culture Techniques; Cell physiology; Cells; Detection; Dyes; Elements; Engineering; Escherichia coli; Evaluation; Event; Exhibits; Exoribonucleases; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Functional disorder; Genetic Transcription; Goals; Government; Hepatitis B; Hepatitis B virus Pol Protein; Hepatocyte; Human; Individual; Infection; Introns; Laboratories; Life Cycle Stages; Ligase; Ligation; Mammalian Cell; Measurement; Methods; Nature; Noise; Oranges; Permeability; Persons; Physiological; Proteins; RNA; RNA Binding; RNA Ligase (ATP); Reporter; Resistance; Signal Pathway; Signal Transduction; Spinach - dietary; Structure; System; Technology; Testing; Therapeutic; Time; Transducers; Transfer RNA; Viral; Viral Genome; Viral Proteins; Virus Diseases; Virus Replication; Work; aptamer; base; chromophore; circular RNA; design; fluorophore; improved; in vivo; insight; next generation sequencing; novel; novel strategies; nutrient absorption; response; sensor; small molecule; synthetic biology; tool; virus pathogenesis

ABSTRACT Application of real-time RNA-based sensors to study concentrations of metabolites and other physiological events by fluorescence is hindered by inadequate expression of RNA by mammalian cells. We have previously developed RNA-based sensors by combining the dye- activating fluorogenic RNA aptamer, Spinach, with metabolite-binding RNA riboswitch elements and implemented them in E. coli. When expressed in mammalian cells, such RNA-based sensors are not as abundant as in E. coli and lack any fluorescence signal. Recent findings have indicated that metazoan tRNA introns generate stable circular RNAs (circRNA) in vivo that can be detected by fluorescence microscopy when incorporating a Spinach-like aptamer sequence. CircRNAs exhibit longer half-lives than their corresponding linear RNAs, possibly due to their resistance to degradation by endogenous exoribonucleases; however in vivo expression of circRNAs is low. Preliminarily, we have devised a new approach to generating circRNAs endogenously that demonstrates 20-fold higher expression levels than the tRNA-intron-based approach. This technology presents an opportunity to express circRNA-based sensors in mammalian culture for the first time and for a range of applications when combined with existing RNA biotechnology. On this basis, I propose to optimize this circRNA expressing technology for increased concentration to improve the fluorescence signal of circRNA-based sensors and expand overall usefulness. Furthermore, I intend to adapt our approach to linear RNA-based sensors to a circRNA context, considering the associated structural and sequence constraints. Finally, I will design the first circRNA-based sensor in mammalian cells by incorporating previously identified RNA aptamers against the truncated polymerase of the hepatitis B virus (HBV). We will also improve on previous approaches to sensor design by optimizing critical transducer sequences of the sensor in a high-throughput manner using next-generation sequencing. Such an optimized sensor will be the basis for a novel HBV infection reporter that does not require engineering of the HBV genome in ways that alter its normal life cycle, as has been done for 25 years. An HBV reporter system that does not intrinsically change the virus’s replication and infection behavior will allow fundamental discoveries as to the mechanism of HBV pathogenesis.

抽象的 应用基于 RNA 的实时传感器来研究代谢物和其他物质的浓度 RNA 表达不足阻碍了荧光引起的生理事件 我们之前通过结合染料开发了基于 RNA 的传感器。 激活荧光 RNA 适体，菠菜，具有代谢物结合 RNA 核糖开关元件 并在大肠杆菌中实现它们在哺乳动物细胞中表达时，这种基于RNA的方法。 传感器不像大肠杆菌那样丰富，并且缺乏任何荧光信号。 表明后生动物 tRNA 内含子在体内产生稳定的环状 RNA (circRNA) 当掺入类菠菜适体时，可以通过荧光显微镜检测到 CircRNA 比其相应的线性 RNA 表现出更长的半衰期，这可能是由于 然而，它们对内源性核糖核酸外切酶降解的抵抗力； 初步，我们设计了一种生成 circRNA 的新方法。 内源性表达水平比基于 tRNA 内含子的表达水平高 20 倍 该技术提供了表达基于 circRNA 的传感器的机会。 首次将哺乳动物培养与现有技术相结合，实现一系列应用 在此基础上，我建议优化这种circRNA表达技术。 增加浓度以改善基于 circRNA 的传感器的荧光信号 此外，我打算将我们的方法调整为基于线性 RNA 的方法。 考虑到相关的结构和序列限制，将传感器连接到 circRNA 环境。 最后，我将在哺乳动物细胞中设计第一个基于 circRNA 的传感器，将 先前鉴定出针对乙型肝炎病毒截短聚合酶的RNA适体 (HBV)。我们还将通过优化关键来改进以前的传感器设计方法。 使用下一代高通量方式传感器的传感器序列 这种优化的传感器将成为新型 HBV 感染报告基因的基础。 不需要像以前那样以改变其正常生命周期的方式对 HBV 基因组进行改造 乙肝病毒报告系统已经研究了 25 年，该系统不会从本质上改变病毒的性质。 复制和感染行为将使有关机制的基本发现成为可能 乙型肝炎的发病机制。