Spatial Regulation of Biomolecular Interactions with a Switchable Trident-Shaped DNA Nanoactuator.

DNA nanostructures with controllable motions and functions have been used as flexible scaffolds to precisely and spatially organize molecular reactions at the nanoscale. The construction of dynamic DNA nanostructures with site-specifically incorporated functional elements is a critical step toward building nanomachines. Artificial self-assembled DNA nanostructures have also been developed to mimic key biological processes like various small biomolecule- and protein-based functional biochemistry pathways. Here, we report a self-assembled dynamic trident-shaped DNA (TS DNA) nanoactuator, in which biomolecules can be tethered to the three "arms" of the TS DNA nanoactuator. The TS DNA nanoactuator is implemented as the mechanical scaffold for the reconfiguration of fluorescent/quenching molecules and the assembly of gold nanoparticles, which exhibit controlled spatial separation. Furthermore, two enzymes (glucose oxidase and horseradish peroxidase) are attached to the two outer arms of the TS DNA nanoactuator, which show an enhanced cascade reaction efficiency compared to free enzymes. The efficiency of the two-enzyme cascade reaction can be spatially regulated by switching the TS DNA nanoactuator between opened, semiopened, and closed states through adding the "thermodynamic drivers" (fuels or antifuels). This is the first report to precisely modulate the relative position of coupled enzyme with multiple states and only based on one dynamic DNA scaffold. The present TS DNA nanoactuator with multistage conformational transition functionality could be applied as a potential platform to precisely and dynamically control the multienzyme pathways and would broaden the scope of DNA nanostructures in single-molecule biology applications.