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和 有可能成为基于RNA的新型疗法。环状RNA很难合成 凝胶提取后得率低，易提纯。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。这项提议的目标是使核糖核酸合成 就像DNA质粒准备试剂盒一样简单。为了做到这一点，我们将开发两个新的高影响力、市场就绪的套件。 我们的第一个试剂盒是“嵌合体细胞内循环RNA纯化试剂盒”，它使用了我们的“旋风”技术。 用于将RNA表达为一个圆圈。当在大肠杆菌中使用时，旋风技术会导致产生 高水平的RNA是以前在常规线性RNA中看不到的。环状RNA的稳定性使它能够 在大肠杆菌中积累到高水平，并在体外和当转染到 哺乳动物细胞，从而缓解了传统线性RNA的持续降解问题。我们也 开发了一种新的RNA纯化适配子，我们将其加入到我们的圈子中，以获得高纯度的 一个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：低 产率、复杂的酶合成反应、RNA降解和费力的RNA问题 净化。我们还将准备改变tRNA修饰目的的嵌合体RNA标记试剂盒 用于RNA标记的酶。这种由酶和合适的底物组成的配套产品将 显著增强传递生物素、荧光染料、细胞靶向部分和其他有用标记的能力 致RNA。为了开发这些新的商业产品，我们的具体目标是：(1)优化一种用于 高产率环状RNA的合成和纯化，无需体外转录。我们将对大肠杆菌进行优化 细胞系，培养条件，葡聚糖结合和洗脱条件，以及RNA储存条件。我们会 还为试剂盒建立规程、手册、重复性和稳定性条件；(2)开发一种 一种简单高效的RNA标记方法。我们将优化反应标记条件和目标 RNA序列，开发协议，手册，并建立稳定性条件，使我们能够创建 可供用户使用的RNA标记试剂盒；(3)开发一种高效洗脱无标签RNA环的系统 葡聚糖珠。我们将进行中试实验，以测试一种全新的RNA纯化试剂盒，该试剂盒涉及- 小分子引发的珠状RNA环化和洗脱。该系统高度简化， 快速，污染物更少，产生的RNA没有任何亲和力标签。总而言之，这个项目 将导致第一个用于合成环状RNA的试剂盒，环状RNA是一种特别具有挑战性但非常重要的 核糖核酸。这项技术通过我们创新的RNA标记试剂盒进行了扩展，使研究人员能够标记、标记 或跟踪用于不同应用的RNA。这些新试剂盒将显著加快环状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.