Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

Abstract

Nanopore technology offers long, accurate sequencing of an DNA or RNA strand via enzymatic ratcheting of the strand through a nanopore in single nucleotide steps, producing stepwise modulations of the nanopore ion current. In contrast to nucleic acids, their daughter molecules, proteins, have neutral peptide backbones and side chains of varying charges. Further, proteins have stable secondary and higher order structures that obstruct protein linearization required for single file nanopore transport. Here, we describe a general approach for realizing unidirectional transport of proteins through a nanopore that neither requires the protein to be uniformly charged nor a pull from a biological enzyme. At high concentrations of guanidinium chloride, we find fulllength proteins to translocate unidirectionally through an a-hemolysin nanopore in a polymer-based membrane, provided that one of the protein ends is decorated with a short anionic peptide. Molecular dynamics simulations show that such surprisingly steady protein transport is driven by a giant electro-osmotic effect caused by binding of guanidinium cations to the inner surface of the nanopore. We show that ionic current signals produced by protein passage can be used to distinguish two biological proteins and the global orientation of the same protein (N-to-C vs. C-to-N terminus) during the nanopore transport. With the average transport rate of one amino acid per 10 μs, our method may enable direct enzyme-free protein fingerprinting or perhaps even sequencing when combined with a high-speed nanopore reader instrument.

Enzyme-less nanopore detection of post-translational modifications within long polypeptides

“…Recently, simulations have suggested an electroosmotic contribution to the translocation of polypeptides through the wild-type (WT) staphylococcal α-hemolysin (αHL) following electrophoretically assisted threading 18 . The ionic charge selectivity necessary to generate electroosmotic flow was attributed, computationally, to guanidinium cations lining the interior of the pore 18 . Here, we use strong electroosmosis directly attributable to the charged side chains in an engineered αHL pore to capture long underivatized polypeptides and detect modifications within them as they are unfolded and translocated.…”

“…As an alternative to electrophoresis, electroosmotic flow (EOF) within a charge-selective protein nanopore has been shown to modulate the binding and dissociation of neutral small molecules 14 , and promote the trapping of short peptides or folded proteins [15][16][17] . Recently, simulations have suggested an electroosmotic contribution to the translocation of polypeptides through the wild-type (WT) staphylococcal α-hemolysin (αHL) following electrophoretically assisted threading 18 . The ionic charge selectivity necessary to generate electroosmotic flow was attributed, computationally, to guanidinium cations lining the interior of the pore 18 .…”

Discrimination of RNA fiber structures using solid-state nanopores

“…S9 †) suggests that at 99% confidence EM block 1.5-2.9% (75-150 pA) more current in comparison to EOM. To confirm the differences in blockade current signals between EOM and EM, we performed supported vector machine learning analysis [31][32][33] using features described in methods (and ESI: Fig S12 -S14 †). Our analysis discriminates between EOM and EM current signals (Fig.…”

“…The features used in the SVM analysis include: Log 10 duration, and current parameters (event mean, standard deviation, minimum, maximum, and the mean of five equal-duration segments), all of which were divided by the open pore current I 0 for normalization. 33 This particular selection of features was done as an attempt to demonstrate that such basic properties of signature ionic current of these RNA brush samples can provide sufficient information for a non-linear classifier to discriminate among the two samples with good accuracy, even if sufficient separation is not apparent in univariate analyses (histograms) or pairwise comparisons (as shown in ESI: Fig S12 †). All features and the normalized trace of an example event are illustrated in ESI: Fig S13 . † The EM events were randomly sampled down to the same number as EOM, for a balanced selection of 187 events per analyte.…”

A click chemistry amplified nanopore assay for ultrasensitive quantification of HIV-1 p24 antigen in clinical samples

“…To circumvent this issue, more robust liposome membranes, polymer bilayers, or solid-state membranes that can maintain integrity under high voltages are highly desirable [71][72][73][74][75][76] . One promising alternative is to replace the lipid membrane with a copolymer membrane 77 , or make a hybrid system by nesting an α-HL protein into a solid-state nanopore in order to achieve robustness and device compatibility, while maintaining the high analytical performance 78 . Secondly, elevated standard deviations were observed in clinical results as normally seen in previous clinical diagnostics studies 79,80 , especially in the lower p24 range, which may cause false calling of results close to LOD.…”