Bioproduction of ∼10 knt single-stranded DNA for constructing large DNA origami structures

Area of Science:

• Nanotechnology
• Molecular Biology
• Biotechnology

Background:

• Traditional DNA origami structures using M13 scaffolds have limitations in size and functional integration.
• Scaling up DNA origami requires longer single-stranded DNA (ssDNA) scaffolds, presenting challenges in preparation and stable synthesis.

Purpose of the Study:

• To establish an efficient biosynthesis platform for producing long ssDNA (approximately 10 kilobase pairs).
• To utilize the synthesized long ssDNA for constructing large, stable DNA origami nanostructures with high yield.
• To overcome the limitations of existing DNA origami scaffolds for enhanced functionalization and size expansion.

Main Methods:

• Developed a phage-phagemid production system for long ssDNA biosynthesis.
• Optimized synthesis parameters in shake flasks and bioreactors to enhance ssDNA yield.
• Assembled large triangular and rectangular DNA origami structures using the produced long ssDNA scaffolds.

Main Results:

• Achieved an approximately tenfold increase in long ssDNA yield through optimized biosynthesis.
• Successfully constructed large DNA origami structures (e.g., 161 nm triangular, 93 × 115 nm rectangular).
• The new structures offer approximately 100 additional potential functionalization sites compared to M13mp18-based structures.

Conclusions:

• The study presents a scalable method for producing long ssDNA scaffolds, crucial for advanced DNA nanotechnology.
• The developed platform provides a practical foundation for fabricating large DNA origami nanostructures with enhanced functionality.
• This work facilitates the creation of more complex and versatile DNA-based nanomaterials.