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A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

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

Xueqiao Li¹, Libang Wang², Wenna Wu¹

¹Department of Applied Chemistry, School of Chemistry and Chemical Engineering, Yantai University, Yantai 264006, China. tao.zhang@ytu.edu.cn.

|December 4, 2024

Summary

DNA origami crossovers were engineered with spacers to control flexibility. Longer spacers relaxed connections, while complementary segments induced rotation, revealing DNA duplex stability requirements and potential applications in responsive materials and torque sensing.

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Studying DNA Looping by Single-Molecule FRET

Studying DNA Looping by Single-Molecule FRET

Published on: June 28, 2014

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.