I-Corps: RNA-adenylation sequencing for rapid ribosome profiling

I-Corps项目更广泛的影响/商业潜力是开发下一代测序技术，旨在量化核糖体保护的mRNA片段，以更高的分辨率推断蛋白质合成。鉴于个体蛋白质生产在疾病诊断和治疗中的核心作用，拟议的技术旨在通过加速个性化医疗，将实验室连接到床边。所提出的技术很容易适用于培养细胞和通过针活检获得的实体组织，并且有望广泛应用于科学家和医疗保健专业人员。此外，探索细胞内蛋白质生产对疾病进展的翻译能力可能为治疗和预防提供新的策略。这项技术可能使基因检测方法对基础研究和临床诊断产生深远的影响。这个I-Corps项目是基于下一代测序技术rna -腺苷化测序的发展。迄今为止，在生物医学和临床实验室中，监测个体蛋白质生产仍然是一个艰巨的挑战。核糖体分析（Ribo-seq）捕获产生蛋白质的mrna并逐步揭示其翻译。这项工作定义了在亚密码子分辨率下提供转录组中核糖体位置和密度快照的要求。目前的Ribo-seq方法需要大量的起始材料，需要昂贵的设备和试剂，并且耗时。与传统的RNA- seq方法相比，所提出的RNA-腺苷化测序技术显著减少了起始材料（~1 ng RNA）的数量，缩短了文库处理时间（~6小时），提高了足迹分辨率（5'端90%的帧内精度）。所提出的技术可以提高翻译的全基因组评估，在单核苷酸分辨率上具有优越的灵敏度。此外，所提出的技术可能适用于RNA测序技术。该技术可用于实验室和临床环境中广泛的RNA下一代测序。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/commercial potential of this I-Corps project is the development of a next generation sequencing technology that aims to quantify ribosome-protected mRNA fragments, to infer protein synthesis with superior resolution. Given the central role of individual protein production in disease diagnosis and treatment, the proposed technology aims to bridge bench-side to bedside by accelerating personalized medicine. The proposed technology is readily applicable to cells in culture and solid tissues obtained by needle biopsy, and is expected to have application for a wide range of scientists and healthcare professionals. Further, the ability to explore the translation of protein production inside cells towards disease progression may offer novel strategies for treatment and prevention. The technology may enable a genetic testing approach that may have far-reaching impacts on basic research and clinical diagnosis. This I-Corps project is based on the development of an RNA-adenylation sequencing, a next generation sequencing technology. To date, monitoring individual protein production remains a formidable challenge in biomedical and clinical laboratories. Ribosome profiling (Ribo-seq) captures protein-producing mRNAs and reveals their translation step-by-step. This effort defines the requirements to provide a snapshot of ribosome positions and density across the transcriptome at a sub-codon resolution. The current Ribo-seq methodology requires a large amount of starting material, needs expensive equipment and reagents, and is time-consuming. Compared to the conventional Ribo-seq method, the proposed RNA-adenylation sequencing technology dramatically reduces the amount of starting material (~1 ng RNA), shortens the library processing time (~6 hour), and increases the footprint resolution (90% in-frame accuracy of 5' end). The proposed technology may improve genome-wide evaluation of translation with superior sensitivity at single-nucleotide resolution. In addition, the proposed technology may be adapted for RNA sequencing technology. The technology may be used for broad RNA next-generation sequencing in both laboratories 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.