Asymmetric Single-Chain MspA nanopores for electroosmotic stretching and sequencing proteins

用于电渗拉伸和蛋白质测序的不对称单链 MspA 纳米孔

• 批准号: 10646810
• 负责人: Aleksei Aksimentiev
• 金额: $ 199.96万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646810
• 关键词: Adoption; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Aspartate; Buffers; Cell Extracts; Cells; Chlorides; Collaborations; Complex; Computer software; Custom; Cysteine; DNA; DNA sequencing; Data; Data Analyses; Detection; Discrimination; Electrolytes; Engineering; Enzymes; Genomics; Geometry; Goals; Head; Health; Heterogeneity; Immobilization; Individual; Ions; Length; Machine Learning; Mammalian Cell; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Motion; Motor; Mutagenesis; Mycobacterium smegmatis; N-terminal; Nucleic Acids; Oligonucleotides; Peptide Library; Peptide Sequence Determination; Peptides; Polymers; Positioning Attribute; Protein Isoforms; Proteins; Proteomics; Pythons; Reading; Reporting; Reproducibility; Research; Resolution; Running; Sampling; Scanning; Series; Signal Transduction; Site; Stretching; Structure; System; Tail; Technology; Testing; Time; Training; Trypsin; VDAC1 gene; Variant; Vertebral column; constriction; design; driving force; experimental study; falls; guanidinium; helicase; in silico; innovation; monomer; mutant; nanoengineering; nanopore; neutravidin; novel; product development; protein aminoacid sequence; prototype; simulation; single molecule; software development; success; tool; voltage

Project Summary / Abstract Protein identification and/or single-molecule protein sequencing from minute amounts could revolutionize our understanding of health by providing a picture of the molecular state of the cell at the level of its most functional molecules. Through this multi-PI proposal, we will develop prototype tools that employ innovative protein-based nanopores for probing the sequence of individual protein molecules. The protein sequencing tool is based on engineered MspA, a porin from Mycobacterium smegmatis that has been utilized in nanopore- based DNA sequencing. However, our protein detection/sequencing tool will be engineered in the following ways: 1) Each of the protein monomers that comprise the octameric assembly are covalently connected, enabling mutagenesis along this single-chain MspA (scMspA) protein to create a series of mutants with asymmetric constrictions as high-resolution nanopores for reading amino acid sequences (recently demonstrated by Niederweis and Wanunu groups), 2) A DNA-processing enzyme will be used to move short peptides and full-length proteins through the pore by conjugation of these peptides to DNA and ATP-mediated DNA translocation, 3) the full compatibility of our system with denaturing electrolyte conditions during pore experiments will facilitate three critical requirements for high accuracy peptide/protein readout in a sequence- independent manner - protein unfolding, protein threading, and a driving force to stretch the protein so it is pulled taut at the pore (all of these recently demonstrated by Wanunu, Aksimentiev, and Chen groups). These combined innovations, combined with key technological capabilities of the team, will allow us to develop a protein sequencing prototype. We propose to achieve our goals through research in three main aims: 1) We will engineer and test various asymmetric scMspA mutants to optimize signal contrast from similar protein sequences with single amino acid substitutions, 2) we will demonstrate helicase-mediated motion of peptide libraries through scMspA mutants and signal decoding, and 3) we will read subsets of full-length unfolded proteins in a complex sample that contains many proteins, and train a model to recognize this sets based on pure protein samples. For the most promising mutant scMspA we will target >90% accuracy in distinguishing among all peptides/proteins in the sample set. Success in our developed platform will result in adoption and product development in order to revolutionize single-molecule and single-cell proteomics.

项目概要/摘要 微量蛋白质鉴定和/或单分子蛋白质测序可能会彻底改变我们的研究 通过提供细胞最高级水平的分子状态图来了解健康 功能分子。通过这个多 PI 提案，我们将开发采用创新技术的原型工具 基于蛋白质的纳米孔，用于探测单个蛋白质分子的序列。蛋白质测序工具 是基于工程化的MspA，一种来自耻垢分枝杆菌的孔蛋白，已被用于纳米孔- 基于 DNA 测序。然而，我们的蛋白质检测/测序工具将按以下方式设计 方式： 1) 组成八聚体的每个蛋白质单体都是共价连接的， 沿着该单链 MspA (scMspA) 蛋白进行诱变，以产生一系列突变体 不对称收缩作为高分辨率纳米孔用于读取氨基酸序列（最近 由 Niederweis 和 Wanunu 小组证明），2）将使用 DNA 加工酶来缩短距离 通过将这些肽与 DNA 和 ATP 介导的缀合，使肽和全长蛋白质穿过孔 DNA 易位，3）我们的系统与孔隙期间的变性电解质条件完全兼容 实验将促进序列中高精度肽/蛋白质读数的三个关键要求 - 独立的方式——蛋白质展开、蛋白质穿线以及拉伸蛋白质的驱动力 毛孔被拉紧（Wanunu、Aksimentiev 和 Chen 团队最近证明了所有这些）。这些 结合创新，结合团队的关键技术能力，将使我们能够开发出 蛋白质测序原型。我们建议通过三个主要目标的研究来实现我们的目标：1）我们 将设计和测试各种不对称 scMspA 突变体，以优化类似蛋白质的信号对比度 具有单个氨基酸取代的序列，2）我们将证明解旋酶介导的肽运动 通过 scMspA 突变体和信号解码来读取文库，3) 我们将读取全长未折叠的子集 包含许多蛋白质的复杂样本中的蛋白质，并训练模型来识别基于 纯蛋白质样品。对于最有希望的突变体 scMspA，我们的目标是区分准确度 >90% 在样品集中的所有肽/蛋白质中。我们开发的平台的成功将导致采用和 产品开发，以彻底改变单分子和单细胞蛋白质组学。