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Biological nanopores for single-pore sensing have the advantage of size homogeneity, structural reproducibility, and channel amenability. In order to translate this to clinical applications, the functional biological nanopore must be inserted into a stable system for high-throughput analysis. Here we report factors that control the rate of pore insertion into polymer membrane and analyte translocation through the channel of viral DNA packaging motors of Phi29, T3 and T7. The hydrophobicity of aminol or carboxyl terminals and their relation to the analyte translocation were investigated. It was found that both the size and the hydrophobicity of the pore terminus are critical factors for direct membrane insertion. An N-terminus or C-terminus hydrophobic mutation is crucial for governing insertion orientation and subsequent macromolecule translocation due to the one-way traffic property. The N- or C-modification led to two different modes of application. The C-terminal insertion permits translocation of analytes such as peptides to enter the channel through the N terminus, while N-terminus insertion prevents translocation but offers the measurement of gating as a sensing parameter, thus generating a tool for detection of markers. A urokinase-type Plasminogen Activator Receptor (uPAR) binding peptide was fused into the C-terminal of Phi29 nanopore to serve as a probe for uPAR protein detection. The uPAR has proven to be a predictive biomarker in several types of cancer, including breast cancer. With an N-terminal insertion, the binding of the uPAR antigen to individual peptide probe induced discretive steps of current reduction due to the induction of channel gating. The distinctive current signatures enabled us to distinguish uPAR positive and negative tumor cell lines. This finding provides a theoretical basis for a robust biological nanopore sensing system for high-throughput macromolecular sensing and tumor biomarker detection.

Single-molecule long-read DNA sequencing with biological nanopores is fast and high-throughput but suffers reduced accuracy in homonucleotide stretches. We now combine the CsgG nanopore with the 35-residue N-terminal region of its extracellular interaction partner CsgF to produce a dual-constriction pore with improved signal and base-calling accuracy for homopolymer regions. The electron cryo-microscopy structure of CsgG in complex with full-length CsgF shows that the 33 N-terminal residues of CsgF bind inside the β-barrel of the pore, forming a defined second constriction. In complexes of CsgG bound to a 35-residue CsgF constriction peptide, the second constriction is separated from the primary constriction by ~25 A. We find that both constrictions contribute to electrical signal modulation during single-stranded DNA translocation. DNA sequencing using a prototype CsgG–CsgF protein pore with two constrictions improved single-read accuracy by 25 to 70% in homopolymers up to 9 nucleotides long. Equipping a protein nanopore with a second constriction improves sequencing of homopolymer DNA stretches.

Protein post-translational modification (PTM) is crucial to modulate protein interactions and activity in various biological processes. Emerging evidence has revealed PTM patterns participate in the pathology onset and progression of various diseases. Current PTM identification relies mainly on mass spectrometry-based approaches that limit the assessment to the entire protein population in question. Here we report a label-free method for the detection of the single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of phi29 DNA packaging motor, which bears the deletion of 25-amino acids (AA) at the C-terminus or 17-AA at the internal loop of the channel. The mutant channels were used to detect propionylation modification via single-molecule fingerprinting in either the traditional patch-clamp or the portable MinION™ platform of Oxford Nanopore Technologies. Up to 2000 channels are available in the MinION™ Flow Cells. The current signatures and dwell time of individual channels were identified. Peptides with only one propionylation were differentiated. Excitingly, identification of single or multiple modifications on the MinION™ system was achieved. The successful application of PTM differentiation on the MinION™ system represents a significant advance towards developing a label-free and high-throughput detection platform utilizing nanopores for clinical diagnosis based on PTM.

The connector channel of bacteriophage phi29 DNA packaging motor has been inserted into the lipid bilayer membrane and has shown potential for the sensing of DNA, RNA, chemicals, peptides, and antibodies. Properties such as high solubility and large channel size have made phi29 channel an advantageous system for those applications; however, previously studied lipid membranes have short lifetimes, and they are frangible and unstable under voltages higher than 200 mV. Thus, the application of this lipid membrane platform for clinical applications is challenging. Here we report the insertion of the connector into the stable polymer membrane in MinION flow cell that contains 2048 wells for high-throughput sensing by the liposome-polymer fusion process. The successful insertion of phi29 connector was confirmed by a unique gating phenomenon. Peptide translocation through the inserted phi29 connector was also observed, revealing the potential of applying phi29 connector for high-throughput peptide sensing.

Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

Ribonucleic acid sequencing can allow us to monitor the RNAs present in a sample. This enables us to detect the presence and nucleotide sequence of viruses, or to build a picture of how active transcriptional processes are changing – information that is useful for understanding the status and function of a sample. Oxford Nanopore Technologies’ sequencing technology is capable of electronically analysing a sample’s DNA directly, and in real-time. In this manuscript we demonstrate the ability of an array of nanopores to sequence RNA directly, and we apply it to a range of biological situations. Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. There are several potential advantages of this approach over other RNA-seq strategies, including the absence of amplification and reverse transcription biases, the ability to detect nucleotide analogues and the ability to generate full-length, strand-specific RNA sequences. Direct RNA sequencing is a completely new way of analysing the sequence of RNA samples and it will improve the ease and speed of RNA analysis, while yielding richer biological information.

The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6–2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria., Pore-forming toxins act by forming oligomeric pores in lipid membranes. Here the authors report the crystal structure of the lysenin pore, providing insights into the assembly and function of the pore in addition to suggesting that its properties make lysenin potentially well-suited for nanopore sensing applications.

A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications. A protein nanopore with a permanent adaptor molecule can continuously identify unlabelled DNA bases with ∼99.8% accuracy. This level of performance could provide the foundation for the development of nanopore-based DNA sequencing technologies that are faster and less expensive than existing approaches.

Staphylococcal alpha-hemolysin (alphaHL) is a beta barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, alphaHL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of alphaHL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of alphaHL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser(217) --> Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of alphaHL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of alphaHL monomers in solution.

The staphylococcal alpha-hemolysin (alphaHL) and leukocidin (Luk) polypeptides are members of a family of related beta-barrel pore-forming toxins. Upon binding to susceptible cells, alphaHL forms water-filled homoheptameric transmembrane pores. By contrast, Luk pores are formed by two classes of subunit, F and S, rendering a heptameric structure displeasing on symmetry grounds at least. Both the subunit stoichiometry and arrangement within the Luk pore have been contentious issues. Here we use chemical and genetic approaches to show that (1) the predominant, or perhaps the only, form of the Luk pore is an octamer; (2) the subunit stoichiometry is 1:1; and (3) the subunits are arranged in an alternating fashion about a central axis of symmetry, at least when a fused LukS-LukF construct is used. The experimental approaches we have used also open up new avenues for engineering the arrangement of the subunits of beta-barrel pore-forming toxins.

Staphylococcal α-hemolysin (αHL) forms a heptameric pore that features a 14-stranded transmembrane β-barrel. We attempted to force the αHL pore to adopt novel stoichiometries by oligomerizing subunit dimers generated by in vitro transcription and translation of a tandem gene. However, in vitro transcription and translation also produced truncated proteins, monomers, that were preferentially incorporated into oligomers. These oligomers were shown to be functional heptamers by single-channel recording and had a similar mobility to wild-type heptamers in SDS-polyacrylamide gels. Purified full-length subunit dimers were then prepared by using His-tagged protein. Again, single-channel recording showed that oligomers made from these dimers are functional heptamers, implying that one or more subunits are excluded from the central pore. Therefore, the αHL pore resists all structures except those that possess seven subunits immediately surrounding the central axis. Although we were not able to change the stoichiometry of the central pore of αHL by the concatenation of subunits, we extended our findings to prepare pores containing one subunit dimer and five monomers and purified them by SDS-PAGE. Two half-chelating ligands were then installed at adjacent sites, one on each subunit of the dimer. Single-channel recording showed that pores formed from this construct formed complexes with divalent metal ions in a similar fashion to pores containing two half-chelating ligands on the same subunit, confirming that the oligomers had assembled with seven subunits around the central lumen. The ability to incorporate subunit dimers into αHL pores increases the range of structures that can be obtained from engineered protein nanopores.

Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process. Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single-channel electrical recordings can be made from both of the major classes of membrane proteins, alpha-helix bundles and beta barrels, which are represented respectively by a K(+) channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays.

Staphylococcal leukocidin (Luk) and alpha-hemolysin (alphaHL) are members of the same family of beta barrel pore-forming toxins (betaPFTs). Although the alphaHL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N- and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on alphaHL suggest that, although the N termini of betaPFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.

Staphylococcal alpha-hemolysin (alphaHL) is a beta barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, alphaHL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of alphaHL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of alphaHL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser(217) --Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of alphaHL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of alphaHL monomers in solution.

Significant progress has been made in membrane protein engineering over the last 5 years, based largely on the re-design of existing scaffolds. Engineering techniques that have been employed include direct genetic engineering, both covalent and non-covalent modification, unnatural amino acid mutagenesis and total synthesis aided by chemical ligation of unprotected fragments. Combinatorial mutagenesis and directed evolution remain, by contrast, underemployed. Techniques for assembling and purifying heteromeric multisubunit pores have been improved. Progress in the de novo design of channels and pores has been slower. But, we are at the beginning of a new era in membrane protein engineering based on the accelerating acquisition of structural information, a better understanding of molecular motion in membrane proteins, technical improvements in membrane protein refolding and the application of computational approaches developed for soluble proteins. In addition, the next 5 years should see further advances in the applications of engineered channels and pores, notably in therapeutics and sensor technology.

A key to understanding bacterial pathogenicity is the mechanism by which water-soluble protein toxins assemble on cell membranes to form oligomeric bilayer-spanning pores. The recent reconstructions from cryo-electron micrographs of three-dimensional pore and prepore structures of the cholesterol-dependent toxin pneumolysin shed new light on the later steps of the assembly of large toxin pores. © 2005 Nature Publishing Group.