Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures

Area of Science:

• DNA nanotechnology
• Biophysics
• Nanomedicine

Background:

• DNA nanostructures can be modified with cholesterol for membrane interactions.
• Cholesterol modification can lead to aggregation, hindering assembly and function.

Purpose of the Study:

• To quantify and optimize the assembly yield of cholesterol-modified DNA origami nanostructures.
• To identify strategies for reducing cholesterol-induced aggregation.

Main Methods:

• Gel electrophoresis was used to quantify the assembly yield of 2D DNA origami tiles (DOTs) and 3D DNA origami barrels (DOBs).
• Various modification strategies were tested, including cholesterol number, spacing, spacer length, and overhangs.
• Detergent treatments were explored to improve yield.

Main Results:

• Reducing cholesterol number from 6 to 1 maximized DOT assembly yield (2% to 100%).
• Optimizing cholesterol separation, decreasing spacer length, and using ssDNA overhangs improved DOT yield.
• Two-step folding did not improve DOB yield, and detergent treatment led to temporary yield increases.

Conclusions:

• Fundamental guidelines were established to minimize cholesterol-induced aggregation in membrane-interacting DNA origami nanostructures.
• Improved assembly yields of well-formed nanostructures are crucial for advancing nanomedicine and biophysics applications.