A DNA walker based on hairpin-shaped DNA aligner and fueled by nicking endonuclease for sensitive and rapid miRNA analysis.

Background: DNA walker-based strategies have gained significant attention in nucleic acid analysis. However, they face challenges related to balancing design complexity, sequence dependence, and amplification efficiency. Furthermore, most existing DNA walkers rely on walking and lock probes, requiring optimization of various parameters like DNA probe sequence, walking-to-lock probe ratio, lock probe length, etc. to achieve optimal performance. This optimization process is time-consuming and adds complexity to experiments. To enhance the performance and reliability of DNA walker nanomachines, there is a need for a simpler, highly sensitive, and selective alternative strategy.

Results: A sensitive and rapid miRNA analysis strategy named hairpin-shaped DNA aligner and nicking endonuclease-fueled DNA walker (HDA-NE DNA walker) was developed. The HDA-NE DNA walker was constructed by modifying hairpin-shaped DNA aligner (HDA) probe and substrate report (SR) probe on the surface of AuNPs. Under normal conditions, HDA and SR remained stable. However, in the presence of miR-373, HDA underwent a conformational transition to an activated structure to continuously cleave the SR probe on the AuNPs with the assistance of Nt.AlwI nicking endonuclease, resulting in sensitive miRNA detection with a detection limit as low as 0.23 pM. Additionally, the proposed HDA-NE DNA walker exhibited high selectivity in distinguishing miRNAs with single base differences and can effectively analyze miR-373 levels in both normal and breast cancer patient serums.

Significance: The proposed HDA-NE DNA walker system was activated by a conformational change of HDA probe only in the presence of the target miRNA, eliminating the need for a lock probe and without sequence dependence for SR probe. This strategy demonstrated a rapid reaction rate of only 30 min, minimal background noise, and a high signal-to-noise ratio (S/B) compared to capture/lock-based DNA walker. The method is expected to become a powerful tool and play an important role in disease diagnosis and precision therapy.