Simple Single-Legged DNA Walkers at Diffusion-Limited Nanointerfaces of Gold Nanoparticles Driven by a DNA Circuit...

We designed and prepared a single-legged DNA walker that relies on the creation of a simple diffusion-limited nanointerface on a gold nanoparticle (DNA/PEG⁽⁺⁾-GNP) track co-modified with fluorescence-labeled hairpin DNA and poly(ethylene glycol) (PEG) containing a positively charged amino group at one end. The movement of our single-legged DNA walker is driven by an enzyme-free DNA circuit mechanism through cascading toehold mediated DNA displacement reactions (TMDRs) using fuel hairpin DNAs. The acceleration of TMDRs was observed for the DNA/PEG⁽⁺⁾-GNP track through electrostatic interaction between the positively charged track and negatively charged DNAs, resulting in the acceleration of the DNA circuit and amplification of the fluorescence signal. Furthermore, the DNA/PEG⁽⁺⁾-GNP track allowed autonomous and persistent movement of a walker DNA strand on the same GNP track, because the intraparticle DNA circuit occurred preferentially by preventing diffusion of the negatively charged free walker DNA strand from near the positively charged tracks into solution through electrostatic interaction. Based on comparative study of kinetics of TMDRs and DNA walking behaviors, it is to be noted that the DNA/PEG⁽⁺⁾-GNP track showed the fastest DNA circuit reaction (walking rate) and the largest number of steps taken by the walker DNA strand compared to other GNP tracks with varying nanointerfaces that differ in terms of their type of charges (no and negative charges), density of positive charges, and number of hairpin DNAs per GNP track. These facts reveal that the positive charges on the GNP track play an important role in the acceleration of the DNA circuit, as well as the successful walking motion of the single-legged DNA strand. By using the fluorescence signal amplification functions, our single-legged DNA walker could be applied directly and successfully to enzyme-free miRNA-detection systems. The miRNA-detection system provided higher discrimination of other mismatched miRNAs and higher sensitivity (the lowest LOD: 4.0 pM) when compared to other miRNA-detection systems based on other GNP tracks without positive charges. Unlike existing single-legged DNA walkers, our single-legged DNA walkers do not require complex processes, such as immobilization of the walker DNA strand on the tracks and precise adjustment of the sequence of walker DNA. Therefore, our strategy, based on the creation of diffusion-limited nanointerfaces, has enormous potential for the applications of single-legged DNA walkers to biosensors, bioimaging, and computing.