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.

项目概要/摘要 我们的目标是实现单个寡肽分子的大规模并行识别和定量 小样本。这种方法的开发将是对主要技术的补充 大规模蛋白质测序和定量，例如质谱分析，将使常规 分析少量蛋白质以及翻译后修饰 (PTM) 的变化。在其第一个 实施，简化以快速达到概念验证，同时生成 进一步优化所需的物理化学参数，新方法将依赖于 具有短寡核苷酸的有机受体。这些缀合物将进行自组装模板 靶向寡肽以提供特定的指纹。在这里，有机受体，例如酮硼酸盐形成 含胺（赖氨酸）的亚胺、具有疏水侧链的环糊精、1,2-二酮识别 胍（精氨酸）、与咪唑（组氨酸）配位的金属络合物和活性烯烃 与硫醇（半胱氨酸）进行可逆迈克尔加成，都将提供弱侧链特异性 通过短互补稳定可逆双链体形成的相互作用 寡核苷酸。每个下一个串联缀合物将与新展示的寡核苷酸表位结合 先前结合的受体，同时被下一个、近端、溶剂暴露的氨基酸侧链选择。 模板将一一选择缀合物，形成最稳定的复合物。通过连接作为 最后一步，每个寡肽将被有效地反向翻译成线性修饰的 DNA 序列， 可以通过例如纳米孔测序来读取。在该项目的前两年，依靠 单分子光谱方法称为 DNA-PAINT，我们将提供概念验证 该过程的基本步骤按设计进行。在目标 1 中，我们将首先展示与 引导寡核苷酸可以吸引特定的寡核苷酸-有机受体缀合物，其中有机 受体通过与特定氨基酸侧链的相互作用来稳定双链体的形成。我们将 直接测量该受体对短寡核苷酸解离率的影响，然后定位这些 观察寡肽模板的结构，并评估寡肽模板的分辨率（特异性） 我们的方法。在目标 2 中，我们将扩展这些研究，使用相同的 DNA-PAINT 方法来研究三元 复合物，其将由第一个缀合物吸引第二个缀合物形成。在我们的最后演示中， 不同的模型肽将以特定序列为模板，随后通过点击化学进行连接 协议。这一进展将使我们项目的下一阶段成为可能，我们将在其中解决自组装问题 肽和蛋白质混合物的分析背景。