Single molecule oligopeptide fingerprinting based on templated self-assembly of oligonucleotide structures

基于寡核苷酸结构模板化自组装的单分子寡肽指纹识别

• 批准号: 10838153
• 负责人: HENRY HESS
• 金额: $ 9.32万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-06 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10838153
• 关键词: Alkenes; Amines; Amino Acids; Arginine; Base Sequence; Basic Science; Binding; Biomedical Research; Chemistry; Clinical Research; Complex; Cyclodextrins; Cysteine; DNA; DNA Sequence; Development; Epitopes; Fingerprint; Goals; Guanidines; Histidine; Hydrophobicity; Imidazole; Imines; Individual; Ligation; Lysine; Mass Spectrum Analysis; Measures; Methods; Modeling; Nucleic acid sequencing; Oligonucleotides; Oligopeptides; Peptide Sequence Determination; Peptides; Phase; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Proteomics; Protocols documentation; Readability; Resolution; Sampling; Side; Solvents; Specificity; Spectrum Analysis; Structure; Sulfhydryl Compounds; Technology; Time; Translating; Variant; Work; design; diketone; metal complex; nanopore; polypeptide; receptor; receptor binding; self assembly; sequencing platform; single molecule

PROJECT SUMMARY/ABSTRACT Our goal is to enable massively parallel identification and quantification of single oligopeptide molecules in very small samples. The development of such method will be complementary to the mainstay technologies for large-scale protein sequencing and quantitation, such as mass spectrometry, and would enable routine analysis of small amounts of protein as well as variations in posttranslational modification (PTMs). In its first implementation, streamlined for quickly reaching the proof-of-concept, while at the same time generating physicochemical parameters required for further optimization, the new method will rely on conjugates of organic receptors with short oligonucleotides. These conjugates will undergo a self-assembly templated by target oligopeptides to provide specific fingerprints. Here, organic receptors, such as, ketoboronates forming imines with amines (lysines), cyclodextrins hosting hydrophobic side chains, 1,2-diketones recognizing guanidines (arginines), metal complex coordinating with imidazoles (histidines), and activated olefins undergoing reversible Michael additions with thiols (cysteines), will all provide weak side- chain-specific interactions that would stabilize otherwise reversible duplex formations by short complementary oligonucleotides. Each next-in-line conjugate would bind to an oligonucleotide epitope newly displayed by the previously bound receptor while being selected by the next, proximal, solvent- exposed amino-acid side chain. One by one, the template would select conjugates forming the most stable complex. Through ligation as the final step, each oligopeptide would effectively be reverse-translated into linear modified DNA sequences that would be readable by, for example, nanopore sequencing. Over the first two years of the project, relying on the single molecule spectroscopy method called DNA-PAINT, we will provide the proof-of-concept that fundamental steps of this process work as designed. We will first show, in Aim 1, that a peptide conjugated to a guide oligonucleotide can attract a specific oligonucleotide-organic receptor conjugate, with the organic receptor stabilizing duplex formation through interactions with the specific amino acid side chain. We will directly measure the impact that this receptor has on off rates of short oligonucleotides, positioning then these observations into the context of structure of oligopeptide templates, and assessing the resolution (specificity) of our approach. In Aim 2, we will expand these studies, using the same DNA-PAINT approach to study ternary complexes, which will be formed by the first conjugates attracting the second. In our final demonstration, different model peptides will template specific sequences, to be subsequently ligated through a click chemistry protocol. This progress will enable the next phase of our project, in which we will tackle self-assembly in the context of analysis of mixtures of peptides and proteins.

项目摘要/摘要 我们的目标是在非常平行的鉴定和量化单个寡肽分子中的大规模平行鉴定和定量 小样品。这种方法的开发将与主要技术相辅相成 大规模的蛋白质测序和定量，例如质谱法，将启用常规 分析少量蛋白质以及翻译后修饰（PTMS）的变化。在第一个 实施，简化以快速达到概念证明，同时生成 进一步优化所需的理化参数，新方法将依赖于 有机受体，具有短寡核苷酸。这些结合者将经历一个自我组装的模板 靶寡肽可提供特定的指纹。在这里，有机受体，例如，酮烯酸酯形成 带有胺（赖氨酸）的亚胺，固定疏水侧链的环糊精，1,2-二酮识别 鸟根（精氨酸），与咪唑（组胺）协调的金属复合物和活化的烯烃 与硫醇（半胱氨酸）进行可逆的迈克尔添加，都将提供弱的侧链特异性 可以通过简短互补的互动来稳定其他可逆的双工形成 寡核苷酸。每个接下来的缀合物都将结合到新显示的寡核苷酸表位 先前由下一个被绑定的受体选择，由下一个，近端溶剂暴露的氨基酸侧链选择。 模板将一一选择形成最稳定的复合物。通过连接作为 最后一步，每种寡肽将有效地反向翻译成线性修饰的DNA序列， 例如，纳米孔测序将可以阅读。在项目的前两年，依靠 单分子光谱法称为DNA-PAINT，我们将提供概念证明 该过程设计的基本步骤。我们将首先在AIM 1中表明肽结合到一个 引导寡核苷酸可以吸引特定的寡核苷酸和有机受体共轭物和有机 受体通过与特定氨基酸侧链相互作用来稳定双链体。我们将 直接测量该受体对短寡核苷酸的关闭速率的影响，然后将其定位 观察到寡肽模板结构的背景，并评估分辨率（特异性） 我们的方法。在AIM 2中，我们将使用相同的DNA-Paint方法扩展这些研究来研究三元 复合物，将由第一个共轭物形成，吸引第二个结合物。在我们的最后示威中 不同的模型肽将模板特定序列，然后通过点击化学结合 协议。这一进展将使我们项目的下一阶段，在其中我们将在其中解决自我组装 肽和蛋白质混合物分析的背景。