Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection.
Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection.
复制标题
适体功能化界面纳米孔可实现氨基酸特异性肽检测。
DOI：
10.1021/acsnano.3c10679
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发表时间：
2024
期刊：
影响因子：
17.1
通讯作者：
Nakatsuka,Nako
中科院分区：
文献类型：
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作者：
Schlotter,Tilman;Kloter,Tom;Hengsteler,Julian;Yang,Kyungae;Zhan,Lijian;Ragavan,Sujeni;Hu,Haiying;Zhang,Xinyu;Duru,Jens;Vörös,János;Zambelli,Tomaso;Nakatsuka,Nako
基于纳米孔技术的单分子蛋白质组学近年来取得了重大进展。然而，要实现单一氨基酸分辨率的纳米孔传感，必须解决几个瓶颈：以纳米级的精度控制纳米孔的大小和减缓分子移位事件。在这里，我们通过将氨基酸特定的DNA适配子整合到具有动态可调孔径的界面纳米孔中来解决这些挑战。苯丙氨酸适配子被用作概念验证：适配子对苯丙氨酸部分的识别导致特定多肽的保留，减慢了转运速度。重要的是，虽然苯丙氨酸适配子是针对游离氨基酸分离的，但适配子被确定为识别苄基或苯基与肽骨架中的羰基的结合，从而能够与特定的含有苯丙氨酸的多肽结合。我们使用光波导光模谱将适配子和含苯丙氨酸的多肽之间的特异性结合从非特异性相互作用(例如静电和疏水相互作用)中分离出来。适体修饰的界面纳米孔含有苯丙氨酸和结构相似氨基酸(即酪氨酸和色氨酸)的对照多肽。当通过降低外加电压来延长纳米孔内适配子-靶相互作用的持续时间时，观察到具有重复模体的离散离子电流水平。测量信号中的这种重复出现的特征表明，所提出的方法有可能解决氨基酸特定的适体识别问题，这是迈向单分子蛋白质组学的一步。
Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocation events. Herein, we address these challenges by integrating amino acid-specific DNA aptamers into interface nanopores with dynamically tunable pore sizes. A phenylalanine aptamer was used as a proof-of-concept: aptamer recognition of phenylalanine moieties led to the retention of specific peptides, slowing translocation speeds. Importantly, while phenylalanine aptamers were isolated against the free amino acid, the aptamers were determined to recognize the combination of the benzyl or phenyl and the carbonyl group in the peptide backbone, enabling binding to specific phenylalanine-containing peptides. We decoupled specific binding between aptamers and phenylalanine-containing peptides from nonspecific interactions (e.g., electrostatics and hydrophobic interactions) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer–target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step toward single-molecule proteomics.