In-Situ ssDNA Isolation from dsDNA Sources as a Streamlined Pathway to DNA Origami Assembly and Testing

Area of Science:

• Nanotechnology
• Molecular Biology
• Biophysics

Background:

• Scaffolded DNA origami is a key nanoscale tool in biomedical and physical sciences.
• Access to long single-stranded DNA (ssDNA) scaffolds is crucial for DNA origami nanodevices.
• Current methods for ssDNA scaffold production are often inefficient and costly.

Purpose of the Study:

• To develop a rapid and scalable method for isolating ssDNA scaffolds from double-stranded DNA (dsDNA).
• To enable direct folding of DNA origami from dsDNA templates.
• To expand the design possibilities for DNA origami nanostructures.

Main Methods:

• Utilized blocking oligonucleotides to selectively release target ssDNA from dsDNA sources.
• Applied the method to linear and supercoiled dsDNA of varying lengths (769–15,101 nucleotides).
• Demonstrated a one-pot, thermally controlled reaction for direct DNA origami folding from dsDNA using blocking and staple strands.

Main Results:

• Successfully isolated target ssDNA sequences from diverse dsDNA templates.
• Achieved direct folding of DNA origami nanodevices from dsDNA templates in a single reaction.
• Showcased the method's versatility with multi-scaffold and gene-encoding DNA origami structures.

Conclusions:

• The developed method offers a faster, more efficient alternative for obtaining ssDNA scaffolds.
• This approach simplifies DNA origami construction and broadens its applicability.
• Facilitates the creation of advanced DNA origami structures for various scientific fields.