Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking.

Area of Science:

• Nanotechnology
• Biochemistry
• Molecular Biology

Background:

• DNA origami enables precise nanoscale construction.
• Functionalizing DNA origami is crucial for advanced applications.
• Existing methods can be complex or inefficient.

Purpose of the Study:

• To develop a versatile, one-pot cross-linking strategy for DNA origami.
• To covalently attach functional groups to DNA origami structures.
• To enhance the stability and functionality of DNA nanostructures.

Main Methods:

• Utilizing psoralen-labeled triplex-forming oligonucleotides (pso-TFOs) for cross-linking.
• Designing adjacent staple strands with extensions forming hairpin duplexes.
• Employing UVA light irradiation to induce psoralen adducts and covalent attachment.
• Creating "superstaples" through bis-adduct formation for enhanced structural integrity.

Main Results:

• Achieved site-specific attachment of pso-TFOs to DNA origami with ~80% efficiency.
• Demonstrated improved resistance to thermal denaturation and T7 RNA polymerase disassembly.
• Confirmed accessibility of the origami core for DNA-binding proteins via DNase I digestion.
• Showcased a scalable and cost-effective method compatible with existing structures.

Conclusions:

• The developed cross-linking method provides robust functionalization and stabilization of DNA origami.
• This technique offers precise control over functional group placement and enhances nanostructure performance.
• The approach is versatile, efficient, and suitable for various DNA nanostructure applications.