Nanopore Detection Using Supercharged Polypeptide Molecular Carriers

Journal of the American Chemical Society, vol. 145, pp. 6371–6382

Abstract

The analysis at the single-molecule level of proteins and their interactions can provide critical information for understanding biological processes and diseases, particularly for proteins present in biological samples with low copy numbers. Nanopore sensing is an analytical technique that allows label-free detection of single proteins in solution and is ideally suited to applications, such as studying protein-protein interactions, biomarker screening, drug discovery, and even protein sequencing. However, given the current spatiotemporal limitations in protein nanopore sensing, challenges remain in controlling protein translocation through a nanopore and relating protein structures and functions with nanopore readouts. Here, we demonstrate that supercharged unstructured polypeptides (SUPs) can be genetically fused with proteins of interest and used as molecular carriers to facilitate nanopore detection of proteins. We show that cationic SUPs can substantially slow down the translocation of target proteins due to their electrostatic interactions with the nanopore surface. This approach enables the differentiation of individual proteins with different sizes and shapes via characteristic subpeaks in the nanopore current, thus facilitating a viable route to use polypeptide molecular carriers to control molecular transport and as a potential system to study protein-protein interactions at the single-molecule level.

Authors 10

1. Xiaoyi Wang

   Imperial College London

   Affiliation as printed

   Department of Chemistry, Imperial College London, Molecular Science Research Hub, London W12 0BZ, U.K

2. Tina-Marie Thomas Aachen DWI - Leibniz Institute for Interactive Materials Institute of Technical and Macromolecular Chemistry

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI − Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen, Germany

   Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

3. Ren Ren

   Imperial College London

   Affiliation as printed

   Department of Chemistry, Imperial College London, Molecular Science Research Hub, London W12 0BZ, U.K

   Department of Metabolism, Digestion and Reproduction, Imperial College London, London W12 0NN, U.K

4. Yu Jerry Zhou Aachen DWI - Leibniz Institute for Interactive Materials Institute of Technical and Macromolecular Chemistry

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI − Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen, Germany

   Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

5. Peng Zhang

   Chinese Academy of Sciences · Changchun Institute of Applied Chemistry · State Key Laboratory of Rare Earth Resources Utilization

   Affiliation as printed

   State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

6. Jinɡjinɡ Li

   Chinese Academy of Sciences · Changchun Institute of Applied Chemistry · State Key Laboratory of Rare Earth Resources Utilization

   Affiliation as printed

   State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

7. Shenglin Cai

   Imperial College London

   Affiliation as printed

   Department of Chemistry, Imperial College London, Molecular Science Research Hub, London W12 0BZ, U.K

8. Kai Liu

   Tsinghua University

   Affiliation as printed

   Engineering Research Center of Advanced Rare Earth Materials, (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China

9. Aleksandar P. Ivanov corresponding

   Imperial College London

   Affiliation as printed

   Department of Chemistry, Imperial College London, Molecular Science Research Hub, London W12 0BZ, U.K

10. Joshua B. Edel corresponding

    Imperial College London

    Affiliation as printed

    Department of Chemistry, Imperial College London, Molecular Science Research Hub, London W12 0BZ, U.K