A Multi‐Input Molecular Classifier Based on Digital DNA Strand Displacement for Disease Diagnostics

DNA-based molecular computing systems for biomarkers have emerged as powerful tools for intelligent diagnostics. However, with the variety of feature biomarkers expanding, current molecular computing systems suffer from the use of a large number of oligonucleotides and limited encoding capability. Here, the study develops an alternative molecular computing approach termed Digital DNA Strand Displacement (DDSD) which recognizes targets and operates target valence through DNA polymerase-based extension and strand release. DDSD significantly reduced the number of used oligonucleotide species, provided robust molecular classifiers. In clinical blood samples, a 96% accuracy rate is achieved with a DDSD-based binary classifier for distinguishing bacterial and viral infections, a 100% accuracy rate is achieved with a multiclass classifier for identifying pathogen types, surpassing existing classifier systems. Moreover, DDSD can be readily expanded. Cascade DDSD is developed, enabling simultaneous computing of up to 14 valence states with a maximum valence of 25. Multiway junction DDSD is implemented to achieve high-valence computing by compact DNA nanostructures rather than split DNA computing units, reducing the potential leakage. The implementation of DDSD enhances the capability of valence-based intelligent molecular diagnostics and multiplexed biomarker detection.