担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Creating alternative nanopores is crucial for advancing single-molecule methodologies. Peptide nanopores scaffolded by DNA nanostructures have emerged as a promising class of chemically synthesizable nanopores with organized geometries. However, no such nanopores have yet achieved single-molecule detection due to size limitations. Here, we overcome this limitation by employing the alamethicin (ALM) peptide, based on the hypothesis that peptides with looser helix-helix packing are advantageous for constructing large-diameter nanopores. The resulting DNA-ALM nanopores exhibited long-lived open states and conductance values comparable to those of natural protein nanopores. To further enhance pore functionality, we introduced asymmetric electrostatic forces by harnessing the intrinsic helix dipole of ALM, which facilitated pore expansion and improved ion transport efficiency. This modification led to enhanced conductance levels and enabled single-molecule sensing of biomolecules, including DNA and peptides. Overall, this work demonstrates a generalizable strategy for constructing functional, chemically synthesizable nanopores, providing a foundation for programmable nanopore design and next-generation biosensing applications.

DOI： 10.1002/anie.3311099

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bacterial secretion systems (SSs) are increasingly recognized as biological nanopores with potential biotechnological applications. Here, we engineered the transmembrane β-barrel of a trimeric autotransporter adhesin (TAA) secreted by the type Vc SS. The coiled-coil segment that occupies the central lumen of the transmembrane β-barrel of an Acinetobacter TAA, AtaA, was removed to design an open β-barrel pore, termed AtaApore. Polypeptides of AtaApore were produced using a cell-free expression system and reconstituted into lipid membranes. Electrophysiological measurements showed ion channel activity of AtaApore with a median conductance of 0.17 nS. Molecular dynamics simulations revealed ion transport properties, including transient trapping of Cl- ions at a constriction formed by R3597 and R3622. Together, to our knowledge, these results provide the first characterization of a nanopore derived from a TAA secreted by the type Vc SS. AtaApore provides a new scaffold for nanopore engineering and a simplified model for probing the mechanism of the type Vc SS.

DOI： 10.3389/fbioe.2026.1764864

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nanopore sensing using transmembrane proteins holds great potential for single-molecule detection. The creation of pore-forming proteins is an emerging research direction, and one promising strategy is the bottom-up de novo design of nanopores. However, de novo α-helical nanopores with sufficiently large dimensions to accommodate single-molecule sensing have not been previously reported. Here, we designed and constructed α-helical nanopores based on two helix-packing motifs, GX6G and GX3G, which we named FFK and LEK. Electrical recordings revealed that both FFK and LEK form nanopores in lipid membranes with high conductance. FFK exhibited multiple conductance states, whereas LEK formed more monodisperse pores. Although both nanopores assembled via a stepwise monomer-joining mechanism, the assembly and disassembly dynamics of FFK were significantly faster than those of LEK. We further demonstrated the single-molecule sensing capabilities of both pores, showing that they can detect cyclodextrin derivatives, while LEK could additionally sense poly-l-lysine. Overall, these results demonstrate the feasibility of designing functional α-helical nanopores for nanopore sensing technologies.

DOI： 10.1021/acsnano.5c15080

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lipid membranes are fundamental elements of cells, serving as barriers that protect the cell interior from the external environment. DNA nanostructures, which can engineer lipid membranes' signal transduction and substance exchange properties, attract interest for their potential applications in the biomedical field. The interaction between DNA nanostructures and lipid membranes is of scientific and technological interest. Here, we investigate the interaction between DNA nanopores (DNPs) and model supported lipid bilayers (SLBs) using real-time quartz crystal microbalance with energy dissipation monitoring (QCM-D). The long observation time and high temporal resolution enable us to visualize the time course of DNPs' interaction with SLBs, including their tethering and incorporating processes. Benefiting from the ability of QCM-D to obtain adsorbed layers' viscoelasticity profiles, we found that the DNPs with three cholesterol tags aggregate and form a rigid layer on the SLB surface during their tethering step. Moreover, our results reveal that the supporting substrates of SLBs impact the incorporation of DNPs, whereby separating the SLBs from the SiO2 sensor surface with poly(ethylene glycol) (PEG) polymer cushion results in faster incorporation. This study not only sheds light on the behavior of DNPs but also establishes QCM-D as an analytical platform for exploring the interactions of membrane-interacting DNA nanostructures, potentially accelerating advancements in DNA nanotechnology.

DOI： 10.1039/d5nr01299f

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lipid vesicles hold potential as artificial cells in bottom-up synthetic biology, and as tools in drug delivery and biosensing. Transmitting molecular signals is a key function for vesicle-based systems. One strategy to achieve this function is by releasing molecular signals from vesicles through nanopores. Nevertheless, in this strategy, an excess of molecular signals may be required to reach the targets, due to the dispersion of the signals during diffusion. The key to achieving the efficient utilization of signals is to shorten the distance between the sender vesicle and the target. Here, we present a pair of DNA nanopores that can connect and form a direct molecular pathway between vesicles. The nanopores are self-assembled from nine single DNA strands, including six 14-nucleotide single-stranded overhangs as sticky-end segments, enabling them to bind with each other. Incorporating nanopores shortens the distance between different populations of vesicles, allowing less diffusion of molecules into bulk solution. To further reduce the loss of molecules, a DNA nanocap is added to one of the nanopore's openings. The nanocap can be removed through the toehold-mediated DNA strand displacement when the nanopore meets its counterpart. Our DNA nanopores provide a novel molecular transmission tool to lipid vesicles-based systems.

DOI： 10.1039/d4nr01344a

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会議種別：口頭発表（招待・特別）  

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会議種別：口頭発表（招待・特別）  

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会議種別：口頭発表（招待・特別）  

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会議種別：口頭発表（招待・特別）  

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出願番号：特願特願2025-088361  出願日：2025年5月

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