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 实时基于RNA的传感器应用代谢物和其他的浓度 通过荧光的生理事件受到RNA表达不足的阻碍 哺乳动物细胞。我们以前已经通过组合染料来开发基于RNA的传感器 用代谢物结合RNA核糖开关元件激活荧光RNA APATMER菠菜 并在大肠杆菌中实施它们。当在哺乳动物细胞中表达时，这种基于RNA 传感器不如大肠杆菌中的大量丰富，并且缺乏任何荧光信号。最新发现 已经表明，后生tRNA内含子在体内产生稳定的圆形RNA（circrna） 当掺入菠菜样的Apatmer时，可以通过荧光显微镜检测到 顺序。 circrnas暴露的半衰期比相应的线性RNA更长，可能 它们因内源性驱虫核酸酶的抗性而抵抗；但是体内表达 Circrnas的低。从初步开始，我们设计了一种新的方法来产生circrnas 内源性表现出比基于tRNA的表达水平高20倍 方法。该技术为表达基于Circrna的传感器的机会提供了机会 哺乳动物文化首次与现有的一系列应用 RNA生物技术。在此基础上，我建议优化这种表达技术 浓度增加以改善基于CIRCRNA的传感器的荧光信号和 扩大总体用途。此外，我打算适应基于线性RNA的方法 考虑到相关的结构和序列约束的传感器。 最后，我将通过编码在哺乳动物细胞中设计第一个基于CIRCRNA的传感器 先前鉴定出针对乙型肝炎病毒的截短聚合酶的RNA适体 （HBV）。我们还将通过优化关键来改善先前的传感器设计方法 使用下一代以高通量方式以高通量方式的传感器序列 测序。这样的优化传感器将成为新型HBV感染报告基因的基础 不需要以改变其正常生命周期的方式对HBV基因组进行工程化 完成了25年。 HBV记者系统不会内在地改变病毒 复制和感染行为将允许关于机制的基本发现 HBV发病机理。