(Nanowerk Spotlight) Controlling the flow of molecules across cellular membranes is a fundamental process in biology, one that scientists have long sought to replicate and enhance through synthetic means. At the heart of this endeavor are nanopores – tiny channels that regulate the passage of ions, small molecules, and even DNA across membranes. While natural protein nanopores have been repurposed for groundbreaking applications like DNA sequencing, their fixed small sizes limit their versatility, especially for transporting larger molecules such as proteins or drug compounds.
The quest to overcome these limitations has driven researchers to explore synthetic alternatives, with DNA nanotechnology emerging as a promising avenue. The DNA origami technique, which allows precise folding of DNA strands into designed 3D structures, has enabled the creation of artificial nanopores with larger dimensions. However, developing nanopores that can dynamically change size while maintaining stability in a lipid membrane has remained a significant challenge.
Previous attempts often resulted in pores that were either too small for macromolecules, structurally unstable, or incapable of reversible size changes once embedded in a membrane. These limitations have hindered progress in areas such as controlled drug delivery, biomolecule sorting, and the development of artificial cellular systems.
Recent advances in DNA origami design and our understanding of lipid membrane interactions have now paved the way for a breakthrough. Researchers from Delft University of Technology and the Max Planck Institute of Biochemistry have developed a novel DNA origami nanopore that can reversibly switch between three distinct sizes, even when inserted into a lipid membrane. This “MechanoPore” (MP) combines concepts from DNA nanotechnology, mechanical engineering, and synthetic biology to achieve controlled size changes that enable selective transport of differently sized molecules.
Design and working principle of the reconfigurable MechanoPore. a) 3D illustration of the DNA nanopore in the open state (MP-O) when embedded in a lipid membrane. b) Top and c) side view of MP-O. Grey cylinders represent dsDNA, while the grey lines are ssDNA. Yellow diamonds represent schematically the attached cholesterol modifications. d) Reversible conformational changes between 3 states (open, intermediate, and closed) of the MP in response to the addition of trigger strands (blue and magenta for opening and closing strands, respectively) and anti-trigger strands (grey). Inset: trigger mechanism: the fully open state (blue) and the closed state (magenta). (Image: Reproduced from DOI:10.1002/adma.202405104, CC BY)
The team designed their MP with a unique structure that allows for dynamic shape changes. The nanopore consists of four L-shaped subunits arranged in a rhombic configuration. Each subunit is composed of a transmembrane barrel section and a cap that rests on top of the membrane. The key to the MP’s flexibility lies in the incorporation of single-stranded DNA segments between these rigid subunits. These flexible linkers act like hinges, allowing the overall structure to change shape in response to specific triggers.
The MP’s inner diameter can range from approximately 11 nanometers in the closed state to 30 nanometers when fully open. This size range is particularly significant as it spans the dimensions of many biologically relevant molecules, from small proteins to larger macromolecular complexes.
The researchers used advanced imaging techniques, including super-resolution microscopy (DNA-PAINT), to confirm that the MPs could successfully adopt their designed conformations and switch between them. However, the real test came when inserting these large DNA structures into lipid membranes.
Using a technique called continuous Droplet Interface Crossing Encapsulation (cDICE), the team embedded the MPs into giant unilamellar vesicles (GUVs) – artificial cell-like structures. Remarkably, the MPs retained their switching ability within this membrane environment, overcoming the lateral pressure exerted by the lipids.
To demonstrate the functional capability of their nanopores, the researchers used fluorescently labeled dextran molecules of varying sizes as cargo. When the pores were fully open, they allowed passage of dextrans up to 150 kilodaltons in size. The intermediate state permitted only dextrans up to 70 kilodaltons, while the closed state blocked all but the smallest 10 kilodalton dextrans.
The team also demonstrated that these conformational switches could be performed repeatedly, with the MPs maintaining functionality even after multiple cycles of opening and closing. This robustness is crucial for potential real-world applications.
The potential applications of this technology extend far beyond drug delivery and biosensing. Perhaps most excitingly, these controllable nanopores represent a significant advancement in the field of synthetic biology, particularly in the creation of artificial cells with sophisticated membrane functions.
Natural cells have evolved complex systems to regulate what goes in and out of their membranes. With these MechanoPores, we’re taking a big step toward replicating and even enhancing these functions in synthetic systems. This could lead to artificial cells capable of performing tasks that natural cells cannot.
For instance, these nanopores could be used to create artificial cellular compartments that can selectively uptake specific molecules based on environmental cues. This could enable the development of smart drug delivery systems that release their payload only under certain conditions. In more advanced applications, networks of these pores could be used to create complex chemical reaction chambers within artificial cells, potentially leading to new ways of producing pharmaceuticals or other valuable compounds.
Moreover, the ability to control molecular transport with such precision opens up new possibilities for studying cellular processes. Researchers could use these nanopores to investigate how changes in membrane permeability affect cellular behavior, potentially leading to new insights into disease mechanisms or drug resistance.
This work showcases the power of interdisciplinary approaches in nanoscale engineering. By combining principles from DNA nanotechnology, mechanical engineering, and membrane biophysics, the researchers have created a functional nanodevice that pushes the boundaries of what’s possible in controlling matter at the molecular scale.
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