SBIR Phase I: An Easy RNA-Adenylation Method for Deep Sequencing of Picogram RNA Samples with High Resolution

这个小企业创新研究第一阶段项目的更广泛影响是推进个性化医疗，以改善治疗和预防。该项目推进了一项技术，以识别新的癌症特异性抗原，并推进癌症免疫治疗。所提出的技术可以应用于培养细胞和通过针活检获得的实体组织。此外，它可以被设计成一个易于使用的系统，供广泛的科学家和医疗保健专业人员使用。该项目旨在开发一种多功能且具有成本效益的文库构建技术，使各种生物样品的蛋白质翻译监测成为可能。RNA分子的高通量测序需要文库的制备。目前的文库构建方法利用适配器连接到目标RNA分子的5‘和3’端。然而，连接接头耗时且效率低，并且所得到的cDNA文库被交叉连接接头和自连接接头的副产物污染，需要额外的纯化步骤。所提出的技术依赖于一种能够对RNA片段进行5 ‘腺苷化和3 ’聚腺苷化的新型酶系统。为了提高RNA两端腺苷化的效率，设计了一种高酶效率的融合酶。此外，为了减少连接时间和提高效率，将专门设计预定向寡核苷酸接头。本项目的重点是去除蔗糖基离心和拟议技术的自动化，这在核糖体分析中以前没有进行过。提出的技术可以成为在实验室和临床设置广泛的核糖体分析的选择方法。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact of this Small Business Innovation Research Phase I project is to advance personalized medicine for improved treatment and prevention. This project advances a technology to identify novel cancer-specific antigens and advances cancer immunotherapy. The proposed technology can be applied to cells in culture and solid tissues obtained by needle biopsy. Furthermore, it can be engineered into an easy-to-use system for a wide range of scientists and healthcare professionals. The proposed project aims to develop a versatile and cost-effective library construction technology that enables protein translation monitoring on a variety of biological samples. High throughput sequencing of RNA molecules requires the preparation of libraries. Current methods for library construction utilize adaptor ligation to the 5'- and 3'- ends of the target RNA molecules. However, ligation of adaptors is time-consuming and low-efficiency, and the resulting cDNA libraries are contaminated with cross- and self-ligation adaptor byproducts and require additional purification steps. The proposed technology relies on a novel enzyme system capable of 5’ adenylation and 3’ polyadenylation of RNA fragments. To increase the efficiency of RNA adenylation at both ends, a fusion enzyme with high enzymatic efficiency is engineered. Moreover, to reduce ligation time and increase efficiency, pre-orientated oligonucleotide adaptors will be specially designed. This project focuses on the removal of the sucrose-based centrifugation and the automation of the proposed technology, which have not been conducted previously in ribosome profiling. The proposed technology can become a method of choice for broad ribosome profiling in both laboratory and clinical settings.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.