Inspired by the biological processes of molecular recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive analytical tools for individual molecules. In particular, nanopore-based single-molecule DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, lower costs and long reads of these methods. Nanopore applications, however, extend far beyond nucleic acid sequencing. In this Review, we present an overview of the broad applications of nanopores in molecular sensing and sequencing, chemical catalysis and biophysical characterization. We highlight the prospects of applying nanopores for single-protein analysis and sequencing, single-molecule covalent chemistry, clinical sensing applications for single-molecule liquid biopsy, and the use of synthetic biomimetic nanopores as experimental models for natural systems. We suggest that nanopore technologies will continue to be explored to address a number of scientific challenges as control over pore design improves.

While DNA sequencing is now amply available, fast, and inexpensive, protein sequencing remains a tremendous challenge. Nanopores may allow for developing a protein sequencer with single-molecule capabilities. As identification of 20 different amino acids currently presents an unsurmountable challenge, fingerprinting schemes are pursued, in which only a subset of amino acids is labeled and detected. This requires modification of amino acids with chemical structures that generate a distinct nanopore ionic current signal. Here, we use a model peptide and the fragaceatoxin C nanopore to characterize six potential tags for a fingerprinting approach using nanopores. We find that labeled and unlabeled proteins can be clearly distinguished and that sensitive detection is obtained for labels with a spectrum of different physicochemical properties such as mass (427-1275 Da), geometry, charge, and hydrophobicity. Additionally, information about the position of the label along the peptide chain can be obtained from individual current-blockade event features. The results represent an important advance toward the development of a single-molecule protein-fingerprinting device with nanopores.

Post-translational modifications (PTMs) of proteins play key roles in cellular processes. Hence, PTM identification is crucial for elucidating the mechanism of complex cellular processes and disease. Here we present a method for PTM detection at the single-molecule level using FraC biological nanopores. We focus on two major PTMs, phosphorylation and glycosylation, that mutually compete for protein modification sites, an important regulatory process that has been implicated in the pathogenic pathways of many diseases. We show that phosphorylated and glycosylated peptides can be clearly differentiated from nonmodified peptides by differences in the relative current blockade and dwell time in nanopore translocations. Furthermore, we show that these PTM modifications can be mutually differentiated, demonstrating the identification of phosphorylation and glycosylation in a label-free manner. The results represent an important step for the single-molecule, label-free identification of proteoforms, which have tremendous potential for disease diagnosis and cell biology.

Ion channels form the basis of information processing in living cells by facilitating the exchange of electrical signals across and along cellular membranes. Applying the same principles to man-made systems requires the development of synthetic ion channels that can alter their conductance in response to a variety of external manipulations. By combining single-molecule electrical recordings with all-atom molecular dynamics simulations, we here demonstrate a hybrid nanopore system that allows for both a stepwise change of its conductance and a nonlinear current-voltage dependence. The conductance modulation is realized by using a short flexible peptide gate that carries opposite electric charge at its ends. We show that a constant transmembrane bias can position (and, in a later stage, remove) the peptide gate right at the most-sensitive sensing region of a biological nanopore FraC, thus partially blocking its channel and producing a stepwise change in the conductance. Increasing or decreasing the bias while having the peptide gate trapped in the pore stretches or compresses the peptide within the nanopore, thus modulating its conductance in a nonlinear but reproducible manner. We envision a range of applications of this removable-gate nanopore system, e.g. from an element of biological computing circuits to a test bed for probing the elasticity of intrinsically disordered proteins.