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

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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.