The physical architectures of information storage systems often dictate how information is encoded, databases are organized, and files are accessed. Here we show that a simple architecture comprised of a T7 promoter and a single-stranded overhang domain (ss-dsDNA), can unlock dynamic DNA-based information storage with powerful capabilities and advantages. The overhang provides a physical address for accessing specific DNA strands as well as implementing a range of in-storage file operations. It increases theoretical storage densities and capacities by expanding the encodable sequence space and simplifies the computational burden in designing sets of orthogonal file addresses. Meanwhile, the T7 promoter enables repeatable information access by transcribing information from DNA without destroying it. Furthermore, saturation mutagenesis around the T7 promoter and systematic analyses of environmental conditions reveal design criteria that can be used to optimize information access. This simple but powerful ss-dsDNA architecture lays the foundation for information storage with versatile capabilities.

Technological leaps are often driven by key innovations that transform the underlying architectures of systems. Current DNA storage systems largely rely on polymerase chain reaction, which broadly informs how information is encoded, databases are organized, and files are accessed. Here we show that a hybrid 'toehold' DNA structure can unlock a fundamentally different, dynamic DNA-based information storage system architecture with broad advantages.This innovation increases theoretical storage densities and capacities by eliminating non-specific DNA-DNA interactions common in PCR and increasing the encodable sequence space. It also provides a physical handle with which to implement a range of in-storage file operations. Finally, it reads files non-destructively by harnessing the natural role of transcription in accessing information from DNA. This simple but powerful toehold structure lays the foundation for an information storage architecture with versatile capabilities.