SurFlex microscopy: Measuring flexibility of surface-tethered biomolecules

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy

Background:

• Molecular flexibility is crucial for biological functions but difficult to quantify for surface-tethered molecules.
• Existing experimental tools are limited for measuring flexibility in aqueous environments.

Purpose of the Study:

• Introduce SurFlex microscopy, a novel fluorescence anisotropy-based method for quantifying molecular flexibility.
• Assess the impact of DNA sequence and cell surface modifications on molecular flexibility.

Main Methods:

• SurFlex microscopy utilizes fluorescence anisotropy to analyze the rotational diffusion of fluorophores attached to tethered molecules.
• Measures apparent molecular flexibility, accounting for tethering, self-interactions, and buffer conditions.
• Applied to bilayer-tethered single-stranded DNA (ssDNA) and surface glycoproteins on red blood cells.

Main Results:

• DNA sequence significantly influences ssDNA flexibility; random sequences show higher stiffness.
• A pathological DNA sequence associated with Huntington's disease displays unusual flexibility.
• Trypsinization of red blood cells reduces glycan flexibility, indicating cell surface modification effects.

Conclusions:

• SurFlex microscopy is a versatile tool for quantifying molecular flexibility of surface-tethered biomolecules.
• Provides insights into the role of molecular flexibility in biological processes.
• Demonstrates sensitivity to sequence-dependent DNA properties and cell surface alterations.