DNA Origami Framework‐Based Spatial Nanochip for Circular ssDNA Assembly and Data Storage

Abstract A 3D DNA spatial chip (DSC) based on an icosahedral DNA origami framework is introduced to construct customized circular single‐stranded DNA (c‐ssDNA) for data storage. Within the confined space of the DSC, thirty addressable location sequences extending from the framework edges are available for designing circular paths and directing the assembly of a series of information oligonucleotides for efficient ligation. This strategy is verified by constructing c‐ssDNAs from up to 15 fragments to encode two poems (800 and 860 nucleotides). Using orthogonal location sites, both poems are simultaneously assembled within a single DSC and read out together. Rolling circle amplification (RCA) and nanopore sequencing enable complete retrieval of all the above data files. The DSCs with distinct fluorescent labels and capture sequences are further functionalized on their outer surfaces, allowing magnetic bead‐based retrieval and rapid identification of specific datasets from a mixture. Moreover, the DSCs maintain data integrity after storage under various conditions. These findings demonstrate that the 3D DNA spatial chip provides an efficient approach for assembling long c‐ssDNA for data storage, addressing limitations by reducing redundancy, enhancing stability, and enabling multiplexed storage and retrieval.