Next-generation RNA synthesis and labeling kits

摘要：环状RNA是一类重要的RNA，具有基本的细胞功能， 有可能成为新型的基于RNA的治疗剂。环状RNA很难合成 并且由于凝胶提取后的低收率而纯化。解决这些问题可能会对 关于环状RNA的学术和生物技术研究。这项提案的目标是使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、市场就绪的套件。 我们的第一个试剂盒是“Chimerna In-cell环状RNA纯化试剂盒”，它使用了我们的“Tornado”技术 来表达RNA在E.大肠杆菌，龙卷风技术的结果，在生产 在高水平的RNA没有以前看到与传统的线性RNA。环状RNA的稳定性使其能够 在E.并且在体外和当转染到大肠杆菌中时也赋予高稳定性。 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 开发了一种新的RNA纯化适体，我们将其纳入我们的圈子，以达到高纯度， 简单的一步洗脱方案。因此，我们的试剂盒将克服体外转录的问题： 产率、复杂的酶促合成反应、RNA降解和费力的RNA合成问题 洁净.我们还将准备Chimerna RNA标记试剂盒， RNA标记酶。这种包含酶和合适的底物的伴随产物将 显著增强赋予生物素、荧光染料、细胞靶向部分和其它有用标签的能力 RNA。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， 无需体外转录即可高产环状RNA合成和纯化。我们将优化E.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 还建立了配套的方案、手册、再现性和稳定性条件;（2）开发一个 一种简单有效的RNA标记方法。我们将优化反应标记条件， RNA序列，开发协议，手册，并建立稳定的条件，使我们能够创建一个 （3）开发了一种用于高效洗脱无标签RNA环的系统， 葡聚糖珠。我们将进行试点实验，以测试一个全新的RNA纯化试剂盒，涉及- 珠RNA环化和由小分子触发的洗脱。该系统高度简化， 快速，污染物更少，产生的RNA没有任何亲和标签。总体而言，该项目 将导致第一个合成环状RNA的试剂盒，这是一种特别具有挑战性但非常重要的类型， 核糖核酸我们的创新RNA标记试剂盒扩展了这项技术，使研究人员能够标记，标记， 或跟踪RNA以用于不同的应用。这些新的试剂盒将大大加速环状RNA的研究。

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.