Next-generation RNA synthesis and labeling kits

下一代 RNA 合成和标记试剂盒

• 批准号: 10553031
• 负责人: Jacob Litke
• 金额: $ 58.6万
• 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/10553031
• 关键词: 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; GTP-Binding Protein alpha Subunits, Gs; Gel; Generations; Genetic Transcription; Goals; Guide RNA; In Vitro; Label; Laboratories; Mammalian Cell; Manuals; Messenger RNA; Methods; Nucleotides; Phase; Plasmids; Porifera; 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; Transfer RNA; Tube; Untranslated RNA; Work; aptamer; 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 有可能是基于RNA的新型治疗的新型类型。圆形RNA很难合成 并由于凝胶提取后的产量低而纯化。解决这些问题可能是一个主要好处 关于循环RNA的学术和生物技术研究。该提议的目的是使RNA合成 像DNA质粒准备套件一样简单。为此，我们将开发两个新的高影响力，可享受市场的套件。 我们的第一个套件是使用我们的“龙卷风”技术 用于将RNA表示为圆圈。当在大肠杆菌中使用时，龙卷风技术会导致生产 以前在常规线性RNA中没有看到的RNA。圆形RNA的稳定性允许 在大肠杆菌中积累到高水平，并且在体外既赋予高稳定性 哺乳动物细胞，从而减轻常规线性RNA的持续降解问题。我们也是 开发了一种新的RNA纯化播放器，我们将其整合到电路中，以实现高纯度 一个简单的一步洗脱协议。那就是我们的套件将克服体外转录的问题：低 产量，复杂的酶合成反应，RNA降解和实验室RNA问题 纯化。我们还将准备重新修改tRNA修改的Chimerna RNA标记套件 RNA标记的酶。该伴侣产品完成酶和合适的底物，将 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 到RNA。为了开发这些新的商业产品，我们的具体目的是：（1）优化套件 高收益圆形RNA合成和纯化，无体外转录。我们将优化大肠杆菌 细胞系，培养条件，葡聚糖结合和洗脱条件以及RNA储存条件。我们将 还建立了套件的协议，手册，可重复性和稳定条件； （2）开发 简单有效的RNA标记的方法。我们将优化反应标记条件和目标 RNA序列，制定协议，手册并建立稳定条件，使我们能够创建一个 用户准备的RNA标签套件； （3）开发一个系统，以高效地洗脱从中的无标签RNA电路 葡萄珠。我们将执行试点实验，以测试一个全新的RNA纯化套件 小分子触发的珠RNA电路和洗脱。该系统高度简化，并且 快速，甚至更少的污染物，并产生没有任何亲和力标签的RNA。总之，这个项目 将导致第一个用于合成圆形RNA的套件，这是一种特别具有挑战性但非常重要的类型 RNA。我们的创新RNA标签套件扩展了这项技术，使研究人员能够标记，标签， 或跟踪潜水应用程序的RNA。这些新套件将显着加速循环RNA研究。