A Robust and Efficient Method to Purify DNA-Scaffolded Nanostructures by Gravity-Driven Size Exclusion Chromatography

Area of Science:

• Nanotechnology
• Biochemistry
• Materials Science

Background:

• Nucleic acid self-assemblies are versatile nanomaterials with programmable assembly and defined geometry.
• Purifying large quantities of DNA nanostructures and nanocomplexes remains a significant challenge for applications.
• Existing purification methods are often limited in scale or incompatible with functionalized nanostructures.

Purpose of the Study:

• To develop a robust and scalable purification method for DNA nanostructures.
• To overcome the limitations of current purification techniques for DNA-templated nanocomplexes.
• To enable efficient purification of functionalized DNA nanostructures for advanced applications.

Main Methods:

• A Sepharose resin-based size exclusion chromatography method was developed.
• Columns were manually packed in-house and designed for reusability.
• Separation utilized low-pressure gravity flow, eluting larger DNA nanostructures before smaller impurities like single-stranded DNA and proteins.

Main Results:

• The method efficiently purified DNA origami assemblies and protein-immobilized DNA nanostructures.
• Purification yielded approximately 100-1000 μg of DNA nanostructures in under 30 minutes.
• Overall collection yield was 50-70% of the crude preparation mixture, significantly higher than agarose gel electrophoresis.
• Purified nanocomplexes demonstrated enhanced precision in evaluating enzyme functions and antibody-triggered complement protein reactions.

Conclusions:

• Sepharose resin-based size exclusion chromatography offers a scalable and efficient solution for purifying DNA nanostructures.
• This method surpasses traditional techniques in yield, speed, and compatibility with functionalized structures.
• The improved purification enables more precise and reliable applications of DNA nanostructures in biochemical assays and diagnostics.