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

这个i-Corps项目的更广泛的影响/商业潜力是开发下一代测序技术，旨在量化受核糖体保护的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快个性化药物的使用，将床边和床边联系起来。这项拟议的技术很容易应用于培养物中的细胞和通过针刺活组织获得的固体组织，并有望在广泛的科学家和医疗保健专业人员中得到应用。此外，探索细胞内蛋白质生产转化为疾病进展的能力可能为治疗和预防提供新的策略。这项技术可能会使基因测试方法成为可能，对基础研究和临床诊断产生深远影响。这个i-Corps项目是基于下一代测序技术-RNA腺化测序的开发。到目前为止，在生物医学和临床实验室中，监测单个蛋白质的生产仍然是一个艰巨的挑战。核糖体图谱(Ribo-seq)捕获了产生蛋白质的mRNAs，并一步步揭示了它们的翻译。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的快照的要求。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，而且非常耗时。与传统的RIBO-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(~1ngRNA)的量，缩短了文库处理时间(~6h)，并提高了足迹分辨率(5‘端90%的帧内精度)。建议的技术可能会改善基因组范围的翻译评估，在单核苷酸分辨率下具有更高的灵敏度。此外，该技术可能适用于RNA测序技术。该技术可在实验室和临床环境中用于广泛的下一代RNA测序。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.