Endogenous Adenosine Triphosphate-Assisted Three-Dimensional DNA Walker Assembled on Soft Nanoparticles for Intracellular MicroRNA Imaging

DNAwalkers, a sophisticated type of nanomachines, exhibit intelligentapplication in biosensing with high programmability and flexibilitybut usually need additional auxiliary driving force, particularlywhen walking on hard surfaces. Herein, we construct a three-dimensional(3D) DNA walker on the soft surface of DNA nanospheres (DSs) by usinga single-stranded DNA (ssDNA), which is powered by endogenous adenosinetriphosphate (ATP) of live cells, so as to sensitively image microRNA(miRNA) in the tumor microenvironment. When the DS walker enters intolive cells, miR-21, a general overexpressed biomarker in cancer cells,binds with the blocking strand (B), releasing the walking strand (W)and triggering an ATP-propelled walking reaction. The walking of theDS walker then generates an increasing Cy3 fluorescence signal thatindicates the content of miR-21 with about 2.73-fold increase in sensitivityand about 157-fold decrease in the detection limit. Notably, the assemblyof the DS walker on soft nanoparticles needs just an easy hybridizationprocess, which facilitates the operation. Meanwhile, this endogenousATP-powered 3D DNA walker walking on the soft surface performs real-timein situ imaging of miR-21 in live cells, which not only avoids thecomplex cell treatment and signal error induced by additional auxiliaryfactors, but also shows high promise of designing programmable DNAnanomachines.

Automated summary

DNAwalkers are advanced nanomachines that can be used for biosensing with high programmability and flexibility. However, they usually require additional auxiliary driving force, especially on hard surfaces. In this study, a three-dimensional DNA walker was constructed on the soft surface of DNA nanospheres using a single-stranded DNA and powered by endogenous ATP. This DNA walker showed sensitive imaging of miRNA in the tumor microenvironment, with significantly increased sensitivity and decreased detection limit. This approach provides a promising method for designing programmable DNA nanomachines without the need for complex cell treatments or additional auxiliary factors.