Abstract

The ability to discriminate multiple biomolecular signals simultaneously is critical for accurate diagnosis of coinfections and evaluation of the medical environment. Yet, achieving multichannel enumeration within a single recording unit remains a significant challenge. Here, we develop a DNA framework-based positional encoding system, termed the DNA Framework Digital Recorder (DFDR), which enables site-specific discrimination of multiple nucleic acid sequences using a uniform signal reporter. The DFDR is composed of a triangular DNA framework where each edge is site-specifically functionalized with orthogonal probes targeting distinct DNA sequences. We demonstrate that a single DFDR unit can resolve 18 nucleic acid targets simultaneously, 6-fold of the multiplexing capacity over conventional DNA self-assembly-based identification systems. Through temporal control of DNA strand inputs, the DFDR supports sequential and rewritable recording of multiplexed signals. We further validate the system by discriminating 16S rRNA mixtures from clinically relevant respiratory pathogens (Haemophilus, Klebsiella, Staphylococcus, and Lactobacillus). Finally, we demonstrate bacterial identification in real-world samples collected from the hospital environment and natural river water. This work establishes a versatile paradigm for high-resolution tracking of multiplexed molecular information with broad implications for synthetic biology, diagnostics, and information storage.