Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy; Dorothy M. Davis Heart and Lung Research Institute; James Comprehensive Cancer Center; College of Medicine; The Ohio State University, Columbus, OH 43210, USA.
Oxford Nanopore Technologies Ltd, Gosling Building, Edmund Halley Road, Oxford Science Park, Oxford, OX4 4DQ, UK.
Biomater Sci. 2021 Dec 21;10(1):167-177. doi: 10.1039/d1bm01264a.
Biological nanopores for single-pore sensing have the advantage of size homogeneity, structural reproducibility, and channel amenability. In order to translate this to clinical applications, the functional biological nanopore must be inserted into a stable system for high-throughput analysis. Here we report factors that control the rate of pore insertion into polymer membrane and analyte translocation through the channel of viral DNA packaging motors of Phi29, T3 and T7. The hydrophobicity of aminol or carboxyl terminals and their relation to the analyte translocation were investigated. It was found that both the size and the hydrophobicity of the pore terminus are critical factors for direct membrane insertion. An N-terminus or C-terminus hydrophobic mutation is crucial for governing insertion orientation and subsequent macromolecule translocation due to the one-way traffic property. The N- or C-modification led to two different modes of application. The C-terminal insertion permits translocation of analytes such as peptides to enter the channel through the N terminus, while N-terminus insertion prevents translocation but offers the measurement of gating as a sensing parameter, thus generating a tool for detection of markers. A urokinase-type Plasminogen Activator Receptor (uPAR) binding peptide was fused into the C-terminal of Phi29 nanopore to serve as a probe for uPAR protein detection. The uPAR has proven to be a predictive biomarker in several types of cancer, including breast cancer. With an N-terminal insertion, the binding of the uPAR antigen to individual peptide probe induced discretive steps of current reduction due to the induction of channel gating. The distinctive current signatures enabled us to distinguish uPAR positive and negative tumor cell lines. This finding provides a theoretical basis for a robust biological nanopore sensing system for high-throughput macromolecular sensing and tumor biomarker detection.
生物纳米孔具有尺寸均一、结构重现性好、通道易于修饰等优点，有利于将单孔传感技术转化为临床应用。为了将其转化为临床应用，功能化的生物纳米孔必须插入到一个稳定的系统中，以实现高通量分析。本研究报告了控制纳米孔插入聚合物膜的速度以及分析物通过 Phi29、T3 和 T7 噬菌体 DNA 包装马达通道转运的因素。研究了氨基末端或羧基末端的疏水性及其与分析物转运的关系。研究发现，纳米孔末端的大小和疏水性都是直接进行膜插入的关键因素。由于单向流特性，N 末端或 C 末端的疏水性突变对于控制插入方向和随后的大分子转运至关重要。N 或 C 修饰导致了两种不同的应用模式。C 末端插入允许诸如肽的分析物通过 N 末端进入通道，而 N 末端插入则阻止了转运，但提供了门控作为传感参数的测量，从而产生了一种用于检测标记物的工具。将尿激酶型纤溶酶原激活物受体(uPAR)结合肽融合到 Phi29 纳米孔的 C 末端，作为 uPAR 蛋白检测的探针。uPAR 已被证明是几种类型癌症（包括乳腺癌）的预测性生物标志物。通过 N 末端插入，uPAR 抗原与单个肽探针的结合由于通道门控的诱导而引起电流的离散阶跃减少。独特的电流特征使我们能够区分 uPAR 阳性和阴性肿瘤细胞系。这一发现为高通量大分子传感和肿瘤生物标志物检测的稳健生物纳米孔传感系统提供了理论基础。
