Nanopore Direct Single-Molecule Protein Sequencing

纳米孔直接单分子蛋白质测序

• 批准号: 9751935
• 负责人: XIAOHUA HUANG
• 金额: $ 41.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751935
• 关键词: Algorithms; Amino Acid Sequence; Amino Acids; Architecture; Basic Science; Biomedical Research; Buffers; Caliber; Cells; Charge; Consensus; DNA sequencing; Data Set; Derivation procedure; Detergents; Development; Dimensions; Drug Targeting; Electrophoresis; Engineering; Film; Foundations; Genomics; Goals; Hybrids; In Situ; In Vitro; Measurement; Measures; Medical; Methods; Modeling; Molecular Diagnosis; Organism; Peptide Sequence Determination; Periodicity; Physiological; Protein Engineering; Protein translocation; Proteins; Proteome; Proteomics; Recombinant Proteins; Recombinants; Site; Sodium Chloride; Structure; Sulfhydryl Compounds; Technology; Thick; Thinness; Time; Vertebral column; Work; clinical Diagnosis; constriction; cost; density; design; digital; drug development; electric field; human disease; innovation; lithography; nanopore; neural network; personalized health care; polypeptide; scaffold; silicon nitride; single molecule; solid state

PROJECT SUMMARY/ABSTRACT: Proteins are the workhorses of cells and organisms. At present, no technologies are available for the routine proteome-scale sequencing and quantification of this physiologically important class of molecules. In this project, we propose to develop a nanopore technology for direct de novo single-molecule protein sequencing. Analogous to nanopore DNA sequencing, the sequences of protein molecules are determined by electronically measuring the alteration of the ionic current flow by the residues along the linear polypeptides while the fully denatured molecules are electrophoretically translocated one at a time through the nanopores. To realize the technology, we need to overcome three major challenges: (1) engineering of nanopores with dimensions (~1 nm diameter and ~0.5 nm thickness) required to distinguish 20 amino acid residues (~0.38 Å spacing) along the linear polypeptide chain; (2) a method for controlled unidirectional translocation of unfolded polypeptides through the nanopores; (3) algorithms for decoding sequences from current blockage profiles. Through several years of conceptual, theoretical and experimental work, we have found potential solutions to these challenges. First, we have invented a new hybrid solid-state/protein/cyclopeptide nanopore architecture that will enable the engineering of nanopores capable of distinguishing the 20 different amino acid residues for de novo protein sequencing. Second, we have also demonstrated a strategy to impart uniform charge density along the polypeptide chain to enable unidirectional translocation of protein through nanopores. Third, we have developed a strategy that has enabled us to model and compute the current blockages of all 207 (=1.28x109) heptamer combinations of 20 amino acids, and thus the current blockage profiles of any proteins. We have also developed the algorithms to decode amino acid sequences from the computed current blockage profiles. Excitingly, with these recent breakthroughs, we have been able to demonstrate the theoretical feasibility of de novo nanopore protein sequencing. We have shown that 12 amino acid residues can be sequenced with >90% consensus accuracy, 2 residues with >85% accuracy, and the other 6 residue decoded as 3 pairs with >90% accuracy. In this project, we propose to implement these innovative approaches aiming to lay the foundation for the experimental realization of nanopore protein sequencing. The ability to sequence and enumerate proteins will enable routine proteome-scale identification and digital quantification of proteins with the ultimate single-molecule and single-cell sensitivity. The ability to sequence proteins at the single-molecule level will enable routine proteome-scale identification and digital quantification of proteins with the ultimate single- molecule sensitivity. If successful, such a disruptive technology will find broad general applications from basic research, drug target identification and precision clinical diagnosis of human diseases, will have potential to transform many aspects of biomedical research, personalized healthcare and medical practice.

项目摘要/摘要： 蛋白质是细胞和生物体的主力。目前还没有技术可以实现 对这一生理学上重要的分子类别进行常规蛋白质组规模测序和定量。在 在这个项目中，我们建议开发一种用于直接从头单分子蛋白质的纳米孔技术 测序。与纳米孔 DNA 测序类似，蛋白质分子的序列由下式确定： 以电子方式测量线性多肽残基对离子电流的改变 而完全变性的分子则通过纳米孔一次一个地进行电泳移位。 为了实现该技术，我们需要克服三个主要挑战：（1）纳米孔工程 区分 20 个氨基酸残基（~0.38 Å）所需的尺寸（~1 nm 直径和~0.5 nm 厚度） 间隔）沿着线性多肽链； (2) 一种展开的受控单向移位方法 多肽通过纳米孔； (3)用于从当前阻塞概况解码序列的算法。 通过几年的概念、理论和实验工作，我们找到了潜在的解决方案 这些挑战。首先，我们发明了一种新型混合固态/蛋白质/环肽纳米孔结构 这将使纳米孔工程能够区分 20 种不同的氨基酸残基 从头蛋白质测序。其次，我们还展示了一种赋予均匀电荷密度的策略 沿着多肽链，使蛋白质能够通过纳米孔单向易位。第三，我们有 开发了一种策略，使我们能够建模和计算所有 207 (=1.28x109) 的当前阻塞 20 个氨基酸的七聚体组合，以及任何蛋白质的当前阻断特征。我们有 还开发了从计算出的当前阻塞曲线中解码氨基酸序列的算法。 令人兴奋的是，通过最近的这些突破，我们已经能够证明 de 的理论可行性。 novo 纳米孔蛋白质测序。我们已经证明 12 个氨基酸残基的测序率 >90% 一致准确率，2 个残基的准确率 >85%，另外 6 个残基解码为 3 对，准确率 >90% 准确性。在这个项目中，我们建议实施这些创新方法，旨在奠定基础 用于纳米孔蛋白质测序的实验实现。排序和枚举的能力 蛋白质将使蛋白质的常规蛋白质组规模鉴定和数字量化成为可能，并具有最终的结果 单分子和单细胞敏感性。在单分子水平上对蛋白质进行测序的能力将 实现常规蛋白质组规模的蛋白质鉴定和数字量化，并具有最终的单 分子敏感性。如果成功，这种颠覆性技术将在基础技术领域找到广泛的应用。 人类疾病的研究、药物靶点识别和精准临床诊断，将有潜力 改变生物医学研究、个性化医疗保健和医疗实践的许多方面。