Parameter Pool-AssistedCentrifugation Sorter forMultiscale Higher-Order DNA Nanomaterials

Higher-order DNA nanomaterials have emerged as programmable tools for probing biological processes, constructing metamaterials, and manipulating mechanically active nanodevices with the multifunctionality and high-performance attributes. However, their utility is limited by intricate mixtures formed during hierarchical multistage assembly, as standard techniques like gel electrophoresis lack the resolution and applicability needed for precise characterization and enrichment. Thus, it is urgent to develop a sorter that provides high separation resolution, broad scope, and bioactive functionality. Here, we present a versatile and scalable sorting pipeline based on a centrifugation parameter pool capable of distinguishing DNA nanomaterials across multiple scales. By tuning parameters, we achieved high-throughput classification of nanostructures, spanning from DNA tile-based constructs to DNA origami-based assemblies (∼50 MDa, 75,000 bp), surpassing conventional methods. Furthermore, we optimized the separation resolution to less than 78 kDa (∼120 bp) at a large scale by sorting DNA tetrahedron structures using this systematic parameter pool-assisted centrifugation strategy. This sorter maintains the integrity and functionality of bioactive materials, facilitating a seamless transition from assembly to application, allowing for integration with proteins and other components to achieve the fabrication of complex functional materials and programmable molecular machines across interdisciplinary fields within the nanotechnology community.