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：环状RNA是一类重要的RNA，具有基本的细胞功能和 有潜力成为基于 RNA 的新型疗法 环状 RNA 难以合成。 由于凝胶提取后产量低而进行纯化，解决这些问题可能是一个主要好处。 关于环状RNA的学术和生物技术研究该提案的目标是使RNA合成。 就像 DNA 质粒制备试剂盒一样简单 为此，我们将开发两种新的高影响力、可上市的试剂盒。 我们的第一个试剂盒是“Chimerna In-cell circRNA纯化试剂盒”，它使用我们的“Tornado”技术 用于将 RNA 表达为圆圈 当在大肠杆菌中使用时，Tornado 技术会产生 RNA 的高水平是传统线性 RNA 所未见的，环状 RNA 的稳定性使其得以实现。 在大肠杆菌中积累到高水平，并且在体外和转染时也具有高稳定性 哺乳动物细胞，从而减轻传统线性 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圈子中，以实现高纯度 因此，我们的试剂盒将克服体外转录的问题：低。 产量、复杂的酶促合成反应、RNA 降解以及费力的 RNA 问题 我们还将准备 Chimerna RNA 标记试剂盒，以重新利用 tRNA 修饰。 该配套产品包含酶和合适的底物，可用于 RNA 标记。 显着增强赋予生物素、荧光染料、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，我们的具体目标是：（1）优化试剂盒。 我们将优化大肠杆菌，实现高产环状RNA的合成和纯化。 细胞系、培养条件、葡聚糖结合和洗脱条件以及 RNA 储存条件。 (2) 开发一个 我们将优化反应标记条件和目标。 RNA 序列、开发方案、手册并建立稳定性条件，使我们能够创建 用户就绪的 RNA 标记试剂盒；(3) 开发一种高效洗脱无标签 RNA 环的系统； 我们将进行试点实验来测试全新的 RNA 纯化试剂盒，其中包括 - 由小分子触发的珠子 RNA 环化和洗脱该系统高度简化且可靠。 总而言之，该项目产生的 RNA 没有任何亲和标签，且污染物更少。 将产生第一个用于合成环状RNA的试剂盒，这是一种特别具有挑战性但非常重要的类型 RNA。我们创新的 RNA 标记试剂盒扩展了该技术，使研究人员能够标记、标记、 或追踪 RNA 的多种应用，这些新试剂盒将显着加速环状 RNA 研究。