A bipedal-unequivalent three-dimensional DNA walker and its biosensing application

Herein, a bipedal-unequivalent three-dimensional DNA walker is designed and applied to biosensing. It involves three components: (i) DNA three-way junction switch (DTWJS), in which two longer strands containing different sequences are partially complementary serving as bipedal-unequivalent walking strand (B-UWS) and the shortest one serves as blocking strand; (ii) fluorophore-labelled hairpins H1 and H2, co-modified onto gold nanoparticles (AuNPs), serving as track strands; (iii) hairpins H3 and H4, contain sequences complementary to H1 and H2, respectively, serving as fuel strands. By target binding to blocking strand, B-UWS is liberated to hybridize with H1 and H2 in turn. The unfolded H1 and H2 get hybridized with H3 and H4, respectively, accompanying by B-UWS sequential release and fluorescence signal accumulation. Then B-UWS continues to hybridize with another track strands to walk. Compared with unipedal and bipedal-equivalent DNA walkers, this bipedal-unequivalent DNA walker has twice the sustainable operation capability through kinetic and affinity studies. Using let-7a as a model target, it shows a detection limit of 68 pM and satisfactory reproducibility in biological fluids. This DNA walker provides a new sustainable operation mode and will be a potential analytical tool in clinical diagnosis.