Nanopore‐Based Protein Sequencing Using Biopores: Current Achievements and Open Challenges

Abstract

The synergy of life sciences discoveries, biomolecular and protein engineering advances, and groundbreaking nanofabrication technologies, has introduced over the past years the wide use of the nanopore-based investigations of matter at the molecular level. This review focuses on the fundamental principles of α-hemolysin (α-HL) protein-based nanopores, as sensitive investigative tools that combine single-molecule detection with the ability to simultaneously manipulate single molecules, in otherwise complex samples. Herein, there are presented some of the efforts directed to control the capture dynamics and translocation speed of tailored polypeptides through the α-HL nanopore, by harnessing the electro-osmotic flow and nanopore-tweezing influence on individual molecules, which are engineered to resemble macrodipoles. The reported applications of this approach suggest a solution to enhance the temporal resolution of nanopore detection, prove the capability of the system in distinguishing between groups of distinct amino acids from the studied poly peptides, and propose a strategy to translate such single-molecule sensors in devices suitable for polypeptide sequencing at unimolecular level.

The Promise of Nanopore Technology: Advances in the Discrimination of Protein Sequences and Chemical Modifications

“…[51] This limitation can be overcome. [49,51,64,65] The acquisition is performed at 250 kHz sampling rate.…”

“…The authors also describe atomic simulation approaches to control the transport dynamics and to distinguish all 20 amino acids. [49] In this review, we first introduce why DNA decoding is not sufficient for understanding human diseases and cellular functions. We highlight that a single gene can encode a variety of proteins with the proteome complexity via epigenetics and other factors.…”

“…The authors also describe atomic simulation approaches to control the transport dynamics and to distinguish all 20 amino acids. [ 49 ]…”

Application of Solid-State Nanopore in Protein Detection

“…In the work of Ouldali et al, amino acids bound with short polycationic carriers are trapped inside the sensing region of the aerolysin nanopore, thus allowing sufficient time for sensitive measurement [110]. The "nanopore tweezer technology" is an alternative solution [111,112]. By engineering peptides flanked by oppositely charged tails at the N-and C-termini, during translocation, the ends of peptides experience oppositely oriented forces similar to an electrostatic "tug of war".…”

Machine Learning to Improve the Sensing of Biomolecules by Conical Track-Etched Nanopore

“…For the past three decades, single nanopore technology have emerged as single-molecule sensors and offer many practical uses such as long read DNA sequencing [ 1 , 2 ]. This was achieved by engineering biological nanopores combined with biological machines to control the DNA translocation speed [ 3 , 4 , 5 , 6 , 7 ]. Beside sequencing, biological nanopores provide a nice platform to analyze the DNA substructure such as hairpin [ 8 ], the hybridization [ 9 , 10 ], zipping [ 11 ] or the interaction with protein [ 12 ].…”