Next-generation RNA synthesis and labeling kits

下一代 RNA 合成和标记试剂盒

• 批准号: 10693332
• 负责人: Jacob Litke
• 金额: $ 60.1万
• 依托单位: CHIMERNA THERAPEUTICS INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10693332
• 关键词: Acceleration; Acetylgalactosamine; Affinity; Alkynes; Area; Avidity; Binding; Biochemical Reaction; Biomedical Research; Biotechnology; Biotin; Cell Line; Cell physiology; Cells; Chemicals; Companions; Complex; Data; Dextrans; Enzymes; Escherichia coli; Exonuclease; Fluorescent Dyes; Gel; Generations; Genetic Transcription; Goals; Guide RNA; In Vitro; Label; Laboratories; Mammalian Cell; Manuals; Marketing; Messenger RNA; Methods; Nucleotides; Phase; Plasmids; Porifera; Predisposition; Production; Protocols documentation; RNA; RNA Degradation; RNA Sequences; RNA chemical synthesis; RNA purification; Reaction; Reproducibility; Research; Research Personnel; Resistance; Sampling; Site; Structure; System; Technology; Testing; Theophylline; Therapeutic; Time; Tornadoes; Transcript; Transfection; Transfer RNA; Tube; Untranslated RNA; Work; aptamer; chemical synthesis; circular RNA; experimental study; innovation; monomer; next generation; novel; plasmid DNA; small molecule; therapeutic RNA

SUMMARY: Circular RNAs are an important class of RNAs that have fundamental cellular functions and have the potential to be novel types of RNA-based therapeutics. Circular RNAs are difficult to synthesize and purify due to low yields after gel extraction. Resolving these issues could be a major benefit to academic and biotechnology research on circular RNA. The goal of this proposal is to make RNA synthesis as simple as a DNA plasmid prep kit. To do this, we will develop two new high-impact, market-ready kits. Our first kit is the “Chimerna In-cell circular RNA purification kit,” which uses our “Tornado” technology for expressing RNAs as a circle. When used in E. coli, the Tornado technology results in the production of RNA at high levels not previously seen with conventional linear RNAs. The stability of circular RNA allows it to accumulate to high levels in E. coli, and also confers high stability in vitro and when transfected into mammalian cells, thus mitigating the persistent degradation problem of conventional linear RNAs. We also developed a new RNA-purification aptamer which we incorporate into our circles to achieve high purity with a simple one-step elution protocol. Thus, our kit will overcome the problems of in vitro transcription: low yield, complex enzymatic synthesis reaction, RNA degradation, and the problem of laborious RNA purification. We will also prepare the Chimerna RNA labeling kit that repurposes a tRNA-modifying enzyme for RNA labeling. This companion product, comprising the enzyme and suitable substrates, will markedly enhance the ability to impart biotin, fluorescent dyes, cell-targeting moieties, and other useful tags to RNA. In order to develop these new commercial products, our specific aims are: (1) To optimize a kit for high-yield circular RNA synthesis and purification without in vitro transcription. We will optimize the E. coli cell line, culturing conditions, dextran binding and elution conditions, and RNA storage conditions. We will also establish protocols, manuals, reproducibility and stability conditions for kitting; (2) To develop an approach for simple and efficient RNA labeling. We will optimize reaction labeling conditions and target RNA sequences, develop protocols, manuals, and establish stability conditions that will allow us to create a user-ready RNA labeling kit; (3) To develop a system for highly efficient elution of tag-free RNA circles from dextran beads. We will perform pilot experiments to test a completely new RNA purification kit involving on- bead RNA circularization and elution triggered by a small molecule. This system is highly simplified and fast, with even fewer contaminants and produces RNA without any affinity tags. Taken together, this project will result in the first kit for synthesizing circular RNA, an especially challenging but highly important type of RNA. This technology is extended with our innovative RNA labeling kit, enabling researchers to label, tag, or track RNAs for diverse applications. These new kits will markedly accelerate circular RNA research.

摘要：圆形RNA是具有基本细胞函数和的重要RNA类 有可能成为基于RNA的疗法的新型类型。 并因凝胶提取后低的Yieeld而净化。 关于圆形RNA的学术和生物技术研究。 像DNA质粒准备套件一样简单。 我们的第一个套件是使用我们的“龙卷风”技术 对于在大肠杆菌中使用的RNA作为圆圈 以前在常规线性RNA的高水平上的RNA。 在大肠杆菌中积累到高水平，并且同意体外高稳定性 哺乳动物细胞，从而减轻常规线性RNA的持续降解问题。 开发了一种新的RNA纯化适体，我们将其纳入我们的coles中，以与With一起实现高纯度。 一个简单的单步洗脱协议。 Yieeld，复杂的酶促合成反应，RNA降解和费力RNA的问题 纯化。 RNA标记的酶。 明显增强了赋予生物素，荧光染料，靶向细胞靶向部分和其他有用标签的能力 为了开发这些新的商业产品，我们的特定目标是：（1） 高收益的RNA合成和体外转录的纯化。 细胞系，培养条件，葡聚糖结合和洗脱条件以及RNA储存条件 还为套件建立协议，手册，繁殖和稳定条件； 简单和效率的RNA标签的方法。 RNA序列，制定协议，手册并建立稳定条件，使我们屈服于创建一个 用户就绪的RNA标签套件； 葡萄珠我们将进行试验实验 珠RNA循环和由 快速，甚至更少的污染物，并且没有亲和力标签 将导致第一个用于合成圆形RNA的套件，这是一种特别挑战但非常重要的类型 RNA使用我们的创新RNA标签套件扩展，使研究人员能够标记，标签，标签，标签，标签，标签，标签。 或跟踪各种应用程序的RNA。