Long-read single-molecule protein sequencing on an array of unfoldase-coupled nanopores

摘要 我们建议开发一个直接对天然全长蛋白质链进行测序的平台的基础 使用解折叠酶偶联纳米孔阵列技术。原则上，这项技术可以用来识别 蛋白质一级序列，以及原核生物中发现的某些翻译后修饰(PTM) 以及真核细胞，具有单分子分辨率。它是对现有的和其他的基础进步 Edman降解、质谱学、荧光标记等下一代蛋白质组学技术 方法，以及基于免疫亲和力的方法，这些方法在读取长度、吞吐量 灵敏度、标记效率和/或合适的亲和试剂的可用性。完整的纳米孔测序 蛋白质链克服了这些限制，因为~1纳米长的传感器直接与 蛋白质链，因为它是由未折叠酶马达蛋白通过毛孔线性驱动的，表现为 序列特定的离子电流信号。因此，可以对天然蛋白质分子进行完整的序列分析 已实现。这种方法是当前纳米孔测序平台的自然技术扩展，使用 在DNA/RNA测序中，控制核酸链通过纳米孔移动的分子马达。 在资助期间，我们将追求三个具体目标：1)建立控制蛋白质的基线方法 通过使用展开酶马达的纳米孔传感器阵列的转移；2)开发计算和 生物信息学方法将原始纳米孔信号数据转换为蛋白质序列信息(氨基酸 呼叫和PTM检测)；以及3)建立分析天然蛋白质和蛋白质组样本的技术。 我们的调查团队是唯一有资格承担这一项目的人： I)我们率先使用解折叠酶偶联纳米孔传感器分析全长蛋白质链，并 最近展示的牛津纳米孔纳米孔阵列装置可以用来直接检测 多肽链和分解单一氨基酸取代(Nivala)。 Ii)这一应用的合作调查人员已经阐明并精细地表征了酶 通过体外生化、单分子和结构研究了解折叠酶马达活性的机制 (Martin)，并领导了用于核酸测序的纳米孔原始信号分析的开发， 包括RNA直接测序、基因组和转录组范围的修饰碱基检测和组装 使用超长DNA纳米孔读数(Jain)对人类基因组进行研究。 三)合作者将提供获得有利的纳米孔技术平台和专门知识的机会，包括 高度平行的纳米孔传感器阵列和定制的纳米孔蛋白质，并提供自然途径 技术转让(牛津纳米孔)，有助于确定和比较项目成果 传统的分析方法，如蛋白质质谱仪(Guttman)，以及令人信服的建议 该项目(TIMP)的成功执行将使技术应用成为可能。

SUMMARY We propose to develop the foundations of a platform for direct sequencing of native, full-length protein strands using unfoldase-coupled nanopore array technology. In principle, this technology could be used to identify protein primary sequence, in addition to certain post-translational modifications (PTMs) found in prokaryotic and eukaryotic cells, with single-molecule resolution. It is a foundational advance over existing and other next-gen proteomic technologies such as Edman degradation, mass spectrometry, fluorescent label approaches, and immunoaffinity-based methods that suffer from limitations in read length, throughput, sensitivity, labeling efficiency, and/or the availability of suitable affinity reagents. Nanopore sequencing of intact protein strands overcomes these limitations because the ~1 nanometer-long sensor directly interacts with the protein strand as it is linearly-driven through the pore by the unfoldase motor protein, manifesting sequence-specific ionic current signals. Thus, complete sequence analysis of native protein molecules can be achieved. This method is a natural technical extension of current nanopore sequencing platforms that use molecular motors to control movement of nucleic acid strands through nanopores in DNA/RNA sequencing. During the grant period, we will pursue three specific aims: 1) Establish baseline methods of controlled protein translocation through nanopore sensor arrays using unfoldase motors; 2) Develop computational and bioinformatic methods to translate raw nanopore signal data into protein sequence information (amino acid calling and PTM detection); and 3) Establish techniques for analysis of native proteins and proteomic samples. Our team of investigators is uniquely qualified to take on this project: i) We pioneered the analysis of full-length protein strands using unfoldase-coupled nanopore sensors and recently demonstrated that the Oxford Nanopore MinION nanopore array device can be used to directly detect peptide strands and resolve single amino acid substitutions (Nivala). ii) Co-investigators on this application have elucidated and exquisitely characterized the enzymatic mechanisms of unfoldase motor activity through in vitro biochemical, single-molecule, and structural studies (Martin), and have led the development of nanopore raw signal analyses for sequencing of nucleic acids, including direct RNA sequencing, genome and transcriptome-wide detection of modified bases, and assembly of a human genome using ultra-long DNA nanopore reads (Jain). iii) Collaborators will provide access to enabling nanopore technology platforms and expertise, including highly-parallel nanopore sensor arrays and customized nanopore proteins, and offer natural routes to technology transfer (Oxford Nanopore), contribute to characterization and comparison of project results to traditional analysis methods such as protein mass spectrometry (Guttman), and advise on compelling technological applications that will be enabled by successful execution of this project (Timp).