DNA crossover flexibilities upon discrete spacers revealed by single-molecule FRET

Area of Science:

• Biophysics
• Structural Biology
• Materials Science

Background:

• DNA origami is a powerful technique for nanoscale construction.
• Understanding the flexibility of DNA crossovers is crucial for designing complex nanostructures.
• Previous studies have characterized Holliday Junctions, but spacer effects on flexibility remain less explored.

Purpose of the Study:

• To investigate the impact of various spacers on the flexibility of double-stranded crossovers in DNA origami.
• To quantify the inter-structural angles and rotational behavior induced by different spacer designs.
• To determine the minimum base pair requirements for stable intramolecular duplex formation.

Main Methods:

• Utilized single-molecule fluorescence resonance energy transfer (smFRET) to measure flexibility.
• Integrated various types of spacers (zero-base, non-complementary, complementary) into DNA origami crossovers.
• Analyzed inter-structural angles and rotational dynamics as a function of spacer length and complementarity.

Main Results:

• The traditional Holliday Junction with zero-base spacers showed an inter-structural angle of 58.7 degrees, consistent with crystallographic data.
• Non-complementary spacers acted as flexible leashes, with longer spacers leading to more relaxed connections.
• Complementary spacers induced rotation in origami structures, reflecting B-duplex characteristics, with a minimum of 5 base pairs required for stability.

Conclusions:

• Spacer design significantly influences DNA origami crossover flexibility and structural behavior.
• Findings provide insights into DNA duplex stability and mechanical properties.
• The study opens avenues for re-engineering crossovers for applications in responsive materials (shrink-swell) and torque sensing.