Abstract
Macromolecule diffusion in cellular microenvironments dictates the kinetics of biochemical processes, yet inevitably limiting the assembly and operation of biomimetic motors. Herein we program in situ accelerated DNA walkers in diffusion-limited microenvironments such as molecularly crowded solutions and cytoplasm. All DNA components, including single-foot walkers, chemically damaged tracks and calibration elements, are anchored on individual gold nanoparticles. Two endogenous enzymes participating in base repair pathways are used to actuate on-particle walking via a base excision/hydrolyzation coupled reaction. The walkers are in situ driven without requiring external drivers and accelerated several times. They also avoid low-efficiency diffusion/assembly procedures and respond to heterogeneous cellular milieus with calibration function. We further regulated the walking kinetics via DNA densities and sets of enzymes, and demonstrated cytoplasmic behaviors of three kinds of walkers. They were utilized to profile DNA repair pathways and monitor enzyme catalysis in living cells.