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.

摘要 实时RNA传感器在研究代谢物和其他物质浓度中的应用 通过荧光的生理事件被RNA的不充分表达所阻碍， 哺乳动物细胞我们之前已经开发了基于RNA的传感器，通过结合染料- 具有代谢物结合RNA核糖开关元件的激活荧光RNA适体，菠菜 并在E.杆菌当在哺乳动物细胞中表达时， 传感器不像E中那么丰富。coli中，并且没有任何荧光信号。最近的调查结果 已经表明后生动物tRNA内含子在体内产生稳定的环状RNA（circRNA）， 当掺入菠菜样适体时， 顺序CircRNA比其相应的线性RNA表现出更长的半衰期，这可能是由于 它们对内源性核糖核酸外切酶降解的抗性;然而， circRNA的水平很低。首先，我们设计了一种新的方法来产生circRNA， 内源性的，表现出20倍以上的表达水平比基于tRNA内含子的 approach.这项技术提供了一个机会，表达circRNA为基础的传感器， 第一次哺乳动物培养，并与现有的 RNA生物技术。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， 扩大整体效益。此外，我打算调整我们的方法，以线性RNA为基础， 传感器到circRNA上下文，考虑相关的结构和序列约束。 最后，我将设计第一个在哺乳动物细胞中基于circRNA的传感器， 先前鉴定的针对B型肝炎病毒截短聚合酶的RNA适体 （HBV）。我们还将改进以前的方法，以传感器设计，优化关键 传感器的传感器序列以高通量的方式使用下一代 测序这种优化的传感器将是新型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改造HBV基因组， 已经做了25年了。一种HBV报告系统，其本质上不改变病毒的 复制和感染行为将使我们能够从根本上发现 HBV发病机制。