Optimising DNA origami assembly by reducing off-target interactions

Area of Science:

• Nanotechnology
• Molecular Biology
• Biophysics

Background:

• DNA origami allows programmable self-assembly of nucleic acids into nanostructures.
• The role of primary base sequence in DNA origami folding reliability is not fully understood.
• Off-target interactions can lead to kinetic traps and reduced assembly yield.

Purpose of the Study:

• To investigate how scaffold sequence affects DNA origami assembly reliability.
• To develop a computational framework for predicting and minimizing off-target interactions.
• To experimentally validate the impact of scaffold sequence on folding yield and structural uniformity.

Main Methods:

• Developed a multi-objective computational framework to score scaffold sequences based on off-target interactions.
• Identified favorable and unfavorable scaffold regions from biological and synthetic sequences.
• Experimentally tested selected scaffold sequences using atomic force microscopy and single-molecule optical tweezers.

Main Results:

• Scaffold sequences predicted to have fewer off-target interactions consistently yielded higher DNA origami assembly.
• Off-target-prone scaffolds resulted in significantly lower assembly yields, even with complementary staple strands.
• Scaffold variants with reduced off-target interactions produced more mechanically uniform origami structures.

Conclusions:

• Off-target sequence effects are a major determinant of DNA origami folding reliability.
• The developed computational tool can guide the selection of optimal scaffold sequences for any DNA origami design.
• Minimizing off-target interactions enhances both the yield and mechanical uniformity of DNA origami nanostructures.