Structural dynamics of nucleic acids by single-molecule FRET.

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique for studying biological processes at the molecular level.
• smFRET provides nanometer-scale information on the structural dynamics of nucleic acids and proteins under various conditions, surpassing traditional ensemble averaging.
• Individual molecule analysis enables the detection of conformational heterogeneity, distinct dynamical events, and relaxation pathways.

Purpose of the Study:

• To outline protocols for performing smFRET measurements on immobilized nucleic acids.
• To detail methods for both static sample chambers and flow cell setups.
• To provide comprehensive steps for DNA/RNA immobilization, sample chamber, and buffer preparation.

Main Methods:

• Wide-field fluorescence microscopy is employed for smFRET measurements.
• Two distinct protocols are presented: one for static sample chambers and another for flow cells.
• Detailed procedures cover nucleic acid immobilization, sample chamber preparation, and buffer conditions.

Main Results:

• Static smFRET measurements offer simpler setup but are limited to single conditions.
• Flow cell measurements provide enhanced control over buffer conditions and allow ligand introduction.
• The protocols facilitate the study of molecular conformational heterogeneity and dynamics.

Conclusions:

• The described protocols enable detailed investigation of nucleic acid dynamics using smFRET.
• Both static and flow cell methods offer unique advantages for molecular studies.
• These methods are crucial for understanding complex biological processes at the single-molecule level.