Abstract
Mechanically interlocked DNA nanostructures are useful as flexible entities for operating DNA-based nanomachines. Interlocked structures made of double-stranded (ds) DNA components can be constructed by irreversibly threading them through one another to mechanically link them. The interlocked components thus remain bound to one another while still permitting large-amplitude motion about the mechanical bond. The construction of interlocked dsDNA architectures is challenging because it usually involves the synthesis and modification of small dsDNA nanocircles of various sizes, dependent on intrinsically curved DNA. Here we describe the design, generation, purification, and characterization of interlocked dsDNA structures such as catenanes, rotaxanes, and daisy-chain rotaxanes (DCRs). Their construction requires precise control of threading and hybridization of the interlocking components at each step during the assembly process. The protocol details the characterization of these nanostructures with gel electrophoresis and atomic force microscopy (AFM), including acquisition of high-resolution AFM images obtained in intermittent contact mode in liquid. Additional functionality can be conferred on the DNA architectures by incorpora...Continue Reading