The synthesis of life from non-living matter has captivated and divided scientists for centuries. This bold goal aims at unraveling the fundamental principles of life and leveraging its unique features, such as its resilience, sustainability, and ability to evolve. Synthetic life represents more than an academic milestone—it has the potential to revolutionize biotechnology, medicine, and materials science. Although the fields of synthetic biology, systems chemistry, and biophysics have made great strides toward synthetic life, progress has been hindered by social, philosophical, and technical challenges, such as vague goals, misaligned interdisciplinary efforts, and incompletely addressing public and ethical concerns. Our perspective offers a roadmap toward the synthesis of life based on discussions during a 2-week workshop with scientists from around the globe.

Due to its pivotal role as a regulator of nucleocytoplasmic transport, the structure and dynamic gating mechanism of the nuclear pore complex (NPC) is a subject of immense interest. Here, we report key recent advancements discussed at the Selective Transport Control in Biological and Biomimetic Nanopores meeting (Monte Verità, Switzerland, 2024) that gathered NPC experts from a range of disciplines. Novel insights were reported from cutting-edge super-resolution techniques that enable the direct interrogation of the NPC’s dynamic central transporter; computational models that unravel the mechanisms of the selective barrier; and synthetic NPC mimics as valuable in vitro models for delineating NPC permeability and transport dynamics. Altogether, three major insights were highlighted: (i) the presence of dynamically organised nuclear transport pathways within the NPC, (ii) the role of nuclear transport receptors that enrich and reinforce the NPC’s selective permeability barrier, and (iii) the ability of DNA origami nanostructures to mimic aspects of the NPC with unprecedented precision. Overall, the advancements marked a convergence in our understanding of NPC function by unraveling its dynamic gating mechanism at the nanoscale.

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules, as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology are harnessed, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between 3 stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behavior with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand – thereby impacting fields as diverse as drug delivery, biomolecule sorting, and sensing, as well as bottom-up synthetic biology.

Nanopores are versatile single-molecule sensors offering a simple label-free readout with great sensitivity. We recently introduced the nanopore electro-osmotic trap (NEOtrap) which can trap and sense single unmodified proteins for long times. The trapping is achieved by the electro-osmotic flow (EOF) generated from a DNA-origami sphere docked onto the pore, but thermal fluctuations of the origami limited the trapping of small proteins. Here, we use site-specific cholesterol functionalization of the origami sphere to firmly link it to the lipid-coated nanopore. We can lock the origami in either a vertical or horizontal orientation which strongly modulates the EOF. The optimized EOF greatly enhances the trapping capacity, yielding reduced noise, reduced measurement heterogeneity, an increased capture rate, and 100-fold extended observation times. We demonstrate the trapping of a variety of single proteins, including small ones down to 14 kDa. The cholesterol functionalization significantly expands the application range of the NEOtrap technology.