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： 蛋白质是细胞和组织的工作主场。目前，没有技术可用于 常规的蛋白质组规模测序和这种物理上重要的分子类别的定量。在 这个项目，我们建议开发直接从头单分子蛋白的纳米孔技术 测序。类似于纳米孔DNA测序，蛋白质分子的序列由 通过电子测量沿线性多肽的残差的离子电流的改变 而完全变性的分子一次通过纳米孔进行电泳一个易位。 要实现这项技术，我们需要克服三个主要挑战：（1）纳米孔的工程 区分20个氨基酸残基所需的尺寸（直径约1 nm，厚度〜0.5 nm）（〜0.38Å 沿线多肽链的间距）； （2）一种控制的单向易位方法 多肽通过纳米孔； （3）用于解码序列的算法。 通过几年的概念性，理论和实验性工作，我们找到了潜在的解决方案 这些挑战。首先，我们发明了一种新的混合固态/蛋白质/环肽纳米孔建筑 这将使能够区分20种不同氨基酸残基的纳米孔的工程 从头蛋白测序。其次，我们还展示了一种赋予均匀电荷密度的策略 沿多肽链，使蛋白质通过纳米孔的单向易位。第三，我们有 制定了一种策略，使我们能够建模和计算所有207的当前障碍（= 1.28x109） 20个氨基酸的七聚体组合，因此所有蛋白质的当前阻塞曲线。我们有 还开发了从计算的电流阻塞曲线中解码氨基酸序列的算法。 令人兴奋的是，随着这些最近的突破，我们能够证明DE的理论可行性 Novo纳米孔蛋白测序。我们已经表明，可以用> 90％测序12个氨基酸残基 共识准确性，2保留> 85％的精度，其他6个保留为3对，> 90％ 准确性。在这个项目中，我们建议实施这些创新的方法旨在奠定基础 为了实现纳米孔蛋白测序。序列和枚举的能力 蛋白质将实现常规蛋白质组规模的识别和对蛋白质的数字定量 单分子和单细胞灵敏度。在单分子水平上测序蛋白质的能力将 启用常规蛋白质组规模鉴定和对蛋白质的数字定量 分子灵敏度。如果成功，这样的破坏性技术将从基本中找到广泛的一般应用 研究，药物靶标识别和对人类疾病的精确临床诊断，将有可能 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。