Building membrane nanopores

Membrane nanopores—hollow nanoscale barrels that puncture biological or synthetic membranes—have become powerful tools in chemical- and biosensing, and have achieved notable success in portable DNA sequencing. The pores can be self-assembled from a variety of materials, including proteins, peptides, synthetic organic compounds and, more recently, DNA. But which building material is best for which application, and what is the relationship between pore structure and function? In this Review, I critically compare the characteristics of the different building materials, and explore the influence of the building material on pore structure, dynamics and function. I also discuss the future challenges of developing nanopore technology, and consider what the next-generation of nanopore structures could be and where further practical applications might emerge

Rebuilding research

The COVID-19 pandemic has had a dramatic impact on the way we do research. Here, I share an approach to rebuild research capacity in a new collaborative fashion termed ‘teamlets’. Teamlets enable a team-based approach to boost morale, increase data integrity, faciliate interdisciplinarity and ensure continuity of expertise

Nanopore-Based Electrical and Label-Free Sensing of Enzyme Activity in Blood Serum

A generic strategy to expand the analytical scope of electrical nanopore sensing is presented. We specifically and electrically detect the activity of a diagnostically relevant hydrolytic enzyme and remove the analytically harmful interference from the biochemically complex sample matrix of blood serum. Our strategy is demonstrated at the example of the renin protease which is involved in regulation of blood pressure. The analysis scheme exploits a new approach to reduce sample complexity while generating a specific read-out signal. Within a single spin-column (i), the protease cleaves a resin-tethered peptide substrate (ii) which is affinity-purified using the same multifunctional resin to remove interfering blood serum components, followed by (iii) detecting the peptide via electrical nanopore recordings. Our approach is beneficial in several ways. First, by eliminating serum components, we overcome limitations of nanopore sensing when challenging samples lead to membrane instability and a poor signal-to-noise ratio. Second, the label-free sensing avoids drawbacks of currently used radiolabel-immunoassays for renin. Finally, the strategy of simultaneous generation and purification of a signal peptide within a multifunctional resin can very likely be expanded to other hydrolytic enzymes dissolved in any analyte matrix and exploited for analytical read-out methods other than nanopore sensing

Nanopores and Nanochannels: From Gene Sequencing to Genome Mapping

DNA strands can be analyzed at the single-molecule level by isolating them inside nanoscale holes. The strategy is used for the label-free and portable sequencing with nanopores. Nanochannels can also be applied to map genomes with high resolution, as shown by Jeffet et al. in this issue of ACS Nano. Here, we compare the two strategies in terms of biophysical similarities and differences and describe that both are complementary and can improve the DNA analysis for genomic research and diagnostics

Reading amino acids in a nanopore

In a step toward nanopore sequencing of proteins, an aerolysin pore discriminates many of the proteinogenic amino acids

Diene-modified nucleotides for the DielsAlder-mediated functional tagging of DNA

We explore the potential of the DielsAlder cycloaddition for the functional tagging of DNA strands. A deoxyuridine triphosphate derivative carrying a diene at position 5 of the pyrimidine base was synthesized using a two-step procedure. The derivative was efficiently accepted as substrate in enzymatic polymerization assays. Diene carrying strands underwent successful cycloaddition with maleimide-terminated fluorescence dyes and a polymeric reagent. Furthermore, a nucleotide carrying a peptide via a DielsAlder cyclohexene linkage was prepared and sequence-specifically incorporated into DNA. The DielsAlder reaction presents a number of positive attributes such as good chemoselectivity, water compatibility, high-yield under mild conditions and no additional reagents apart from a diene and a dienophile. Furthermore, suitable dienophiles are commercially available in the form of maleimide-derivatives of fluorescent dyes and bioaffinity tags. Based on these advantages, diene- and cyclohexene-based nucleotide triphosphates are expected to find wider use in the area of nucleic acid chemistry

Nanotechnology: Changing of the guard

Membrane proteins control access of ions and molecules to a cell's interior, shuttle cargo and information across the cell boundary, and determine the cell's shape. Engineering the function of these proteins is key to the development of vaccines, biofuels, biosensor elements, and research tools. However, the range of accessible architectures is limited, because protein engineering usually involves making relatively modest structural changes to existing protein structures; folding extensively altered polypeptides into defined structures is very challenging. Recent studies have shown that some membrane-protein functions can be mimicked with DNA nanostructures, which are easier to manipulate than their natural templates

Gating-like Motions and Wall Porosity in a DNA Nanopore Scaffold Revealed by Molecular Simulations

Recently developed synthetic membrane pores composed of folded DNA enrich the current range of natural and engineered protein pores and of nonbiogenic channels. Here we report all-atom molecular dynamics simulations of a DNA nanotube (DNT) pore scaffold to gain fundamental insight into its atomic structure, dynamics, and interactions with ions and water. Our multiple simulations of models of DNTs that are composed of a six-duplex bundle lead to a coherent description. The central tube lumen adopts a cylindrical shape while the mouth regions at the two DNT openings undergo gating-like motions which provide a possible molecular explanation of a lower conductance state observed in our previous experimental study on a membrane-spanning version of the DNT (ACS Nano 2015, 9, 1117-26). Similarly, the central nanotube lumen is filled with water and ions characterized by bulk diffusion coefficients while the gating regions exhibit temporal fluctuations in their aqueous volume. We furthermore observe that the porous nature of the walls allows lateral leakage of ions and water. This study will benefit rational design of DNA nanopores of enhanced stability of relevance for sensing applications, of nanodevices with tunable gating properties that mimic gated ion channels, or of nanopores featuring defined permeation behavior

Nanopores: synergy from DNA sequencing to industrial filtration - small holes with big impact

Nanopores in thin membranes play important roles in science and industry. Single nanopores have provided a step-change in portable DNA sequencing and understanding nanoscale transport while multipore membranes facilitate food processing and purification of water and medicine. Despite the unifying use of nanopores, the fields of single nanopores and multipore membranes differ - to varying degrees - in terms of materials, fabrication, analysis, and applications. Such a partial disconnect hinders scientific progress as important challenges are best resolved together. This Viewpoint suggests how synergistic crosstalk between the two fields can provide considerable mutual benefits in fundamental understanding and the development of advanced membranes. We first describe the main differences including the atomistic definition of single pores compared to the less defined conduits in multipore membranes. We then outline steps to improve communication between the two fields such as harmonizing measurements and modelling of transport and selectivity. The resulting insight is expected to improve the rational design of porous membranes. The Viewpoint concludes with an outlook of other developments that can be best achieved by collaboration across the two fields to advance the understanding of transport in nanopores and create next-generation porous membranes tailored for sensing, filtration, and other applications