Driven magnetic 3D DNA walker coupled with CHA based on light-activatable hairpin: A dual-amplification strategy for ultrasensitive hantavirus RNA sensing.

BACKGROUND: Orthohantavirus dabieshanense (DBSV) is a newly emerging hantavirus and poses a potential threat to human health. Current detection methods based on polymerase chain reaction (PCR) assays for other hantaviruses such as Hantaan virus (HTNV) and Seoul virus (SEOV) may carry the risk of false negatives. This study aims to address the technical bottlenecks of low accuracy and insufficient sensitivity in current detection of DBSV.

RESULTS: We developed a signal amplification strategy mediated by a photoactivated magnetic 3D DNA walker, which enables light-controllable fluorescence detection. Specifically, this fluorescence sensing platform introduces the catalytic hairpin assembly (CHA) technique for dual signal amplification. This system requires dual activation by UV irradiation and target RNA. Then, exonuclease III (Exo III) provides propulsion for the walking process to release a specific trigger strand, subsequently activating the CHA cycle and producing a fluorescent signal. Experimental results confirm that the system displays excellent selectivity and anti-interference ability, successfully distinguishing target sequences with single-base mismatch (SBM), double-base mismatch (DBM), and triple-base mismatch (TBM). Furthermore, the platform achieves a DBSV-RNA detection limit (LOD) of 30 fM with a linear range spanning 10-1-106 pM. The applicability of the method has been rigorously validated using human serum specimens.

SIGNIFICANCE: This study provides a novel strategy for the application of spatiotemporally controllable DNA walkers in RNA detection, demonstrating considerable potential for early viral diagnosis and field monitoring.