Engineering the mechanosensitivity of single DNA molecules via high-throughput microfluidic force spectroscopy

Area of Science:

• Biophysics
• Molecular Biology
• Materials Science

Background:

• Single-molecule force spectroscopy (SMFS) is crucial for understanding biomolecular mechanics.
• Current SMFS methods have limited throughput, hindering comprehensive sequence analysis.
• Investigating the mechanical properties of DNA structures is vital for molecular biology.

Purpose of the Study:

• To develop a high-throughput assay for measuring biomolecular responses to mechanical force.
• To enable systematic sequence-function mapping of DNA structures under force.
• To explore the relationship between kinetic stability and mechanical fragility in DNA.

Main Methods:

• Development of a single-molecule, multiplexed, microfluidic force spectroscopy (SM³FS) assay.
• Parallelized microfluidics enabling measurement of up to 80 sequence variants per experiment.
• Stretching, overstretching, and unzipping of 241 DNA structures, generating over 130,000 single-molecule traces.

Main Results:

• Achieved unprecedented sequence throughput in force spectroscopy.
• Identified DNA structures exhibiting kinetic stability coupled with mechanical fragility (rupture force < 3 pN).
• Demonstrated that mechanosensitivity can be an intrinsic property of multivalent biomolecular systems.

Conclusions:

• The SM³FS assay significantly enhances the potential of force spectroscopy for biomolecular studies.
• Revealed novel insights into the mechanical properties and mechanosensitivity of DNA structures.
• Opens avenues for high-throughput nonequilibrium studies of biomolecules.