Design, assembly, characterization, and operation of double-stranded interlocked DNA nanostructures

Area of Science:

• Nanotechnology and Molecular Engineering
• Biomolecular Self-Assembly
• DNA Nanostructures

Background:

• Mechanically interlocked DNA nanostructures are crucial for developing DNA-based nanomachines.
• Existing methods for constructing these interlocked double-stranded (ds) DNA architectures are challenging, often requiring intrinsically curved DNA and complex synthesis.
• The ability to create flexible, mechanically linked DNA components is key to advancing nanoscale device functionality.

Purpose of the Study:

• To describe a protocol for the design, generation, purification, and characterization of interlocked dsDNA structures, including catenanes, rotaxanes, and daisy-chain rotaxanes (DCRs).
• To demonstrate precise control over threading and hybridization for constructing these complex nanostructures.
• To showcase methods for characterizing these nanostructures using gel electrophoresis and atomic force microscopy (AFM).

Main Methods:

• Development of a step-by-step protocol for assembling interlocked dsDNA architectures.
• Utilizing precise control of threading and hybridization during the assembly process.
• Characterization via gel electrophoresis and high-resolution liquid-phase AFM imaging.

Main Results:

• Successful design and generation of interlocked dsDNA structures: catenanes, rotaxanes, and DCRs.
• Demonstration of assembly completion times: 2 days for catenanes and 3 days for rotaxanes.
• Characterization confirming the successful formation and structural integrity of the nanostructures.

Conclusions:

• The presented protocol enables the precise construction of mechanically interlocked DNA nanostructures.
• These DNA architectures can be further functionalized with proteins, molecular switches, or fluorophores for advanced applications.
• This work provides a foundation for creating sophisticated DNA-based nanomachines with diverse functionalities.