Engineering Biological Nanopore Approaches toward Protein Sequencing

Abstract

Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while the methods we have for identifying and quantifying individual proteins are still inadequate to perform protein sequencing at the single-molecule level. Nanopore-inspired systems devoted to understanding how single molecules behave have been extensively developed for applications in genome sequencing. These nanopore systems are emerging as prominent tools for protein identification, detection, and analysis, suggesting realistic prospects for novel protein sequencing. This review summarizes recent advances in biological nanopore sensors toward protein sequencing, from the identification of individual amino acids to the controlled translocation of peptides and proteins, with attention focused on device and algorithm development and the delineation of molecular mechanisms with the aid of simulations. Specifically, the review aims to offer recommendations for the advancement of nanopore-based protein sequencing from an engineering perspective, highlighting the need for collaborative efforts across multiple disciplines. These efforts should include chemical conjugation, protein engineering, molecular simulation, machine-learning-assisted identification, and electronic device fabrication to enable practical implementation in real-world scenarios.

Molecular Determinants of Current Blockade Produced by Peptide Transport Through a Nanopore

“…Thus, the force of the electric field alone may not be sufficient to capture and unidirectionally transport a polypeptide chain through a nanopore. Multiple solutions have been described to address this problem, which includes the use of electro-osmotic , or dielectrophoretic forces, or changing the charge of the polypeptide by either bathing a protein in a detergent solution, − changing pH of the solution, and/or appending the proteins with charged tags. ,, Please see recent reviews for a complete account of the literature. ,, …”

Orientational Pathways during Protein Translocation through Polymer-Modified Nanopores

“…A prototypical example is the nuclear pore complex, an intricate molecular architecture that allows for a precise control of the transport of macromolecules between the nucleus and cytoplasm . Other biological nanoconstructs, such as pore-forming toxins, find application in nanopore-based single-molecule sensors. , Controlling protein orientation inside biological nanopores also offers the possibility to engineer nanoreactors for single-molecule studies …”

Sensing PEGylated Peptide Conformations Using a Protein Nanopore

“…Engineered protein pores have been used to sense and characterize various biomolecules such as nucleic acid fragments, oligosaccharides, peptides, and protein segments. − Membrane protein-based nanopores are widely exploited as they can be easily obtained from natural sources with high structural stability. ,,− Their unique geometry and permeability components have been used to build nanopore sensors. ,, Furthermore, they are compatible with studying many biological molecules due to their constriction zone and are flexible to protein engineering to enable high-resolution single-molecule detection. ,− Recently, synthetic DNA and α-helical pores have been introduced due to their sophisticated structure for sensing analytes. − Most nanopore studies have focused on simple small molecules and lengthened polymer chains. ,− Recently, nanopore technology has been used to sense complex biomacromolecules, including peptide and protein segments. − The unstructured peptides can be easily detected using nanopores, whereas larger protein fragments pose challenges due to their intricate folding pattern. ,− ,, In such cases, wide-diameter pores of defined geometry would expand the scope for detecting large folded proteins and complex biopolymers of stable structural conformations. ,, …”