Kinetic FRET Assay to Measure Binding-Induced Conformational Changes of Nucleic Acids

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Nucleic acid (NA) structure and folding are altered by interactions with small molecules and proteins.
• Understanding these molecular mechanisms requires biophysical characterization.
• Real-time kinetic measurements are crucial for elucidating binding and folding dynamics.

Purpose of the Study:

• To introduce kinFRET (kinetics Förster resonance energy transfer), a novel real-time ensemble FRET methodology.
• To measure the binding and folding kinetics of nucleic acids.
• To demonstrate the utility of kinFRET in studying NA-analyte interactions and conformational changes.

Main Methods:

• Development of a chip-based biosensor for real-time Förster resonance energy transfer (FRET) measurements.
• Application of kinFRET to monitor conformational changes of an aptamer (MN19) interacting with small-molecule analytes.
• Characterization of RNA recognition kinetics by tandem K homology (KH) domains of human insulin-like growth factor II mRNA-binding protein 3 (IMP3).

Main Results:

• kinFRET successfully monitored conformational changes in aptamer MN19 upon small-molecule binding.
• Distinct kinetic contributions of two KH domains in IMP3 during RNA recognition were revealed.
• The methodology allows direct observation of conformational dynamics during NA-analyte interactions.

Conclusions:

• kinFRET is a robust method for studying the kinetics of nucleic acid conformational changes.
• The technique provides insights into the molecular mechanisms of NA-analyte interactions.
• kinFRET is well-suited for characterizing binding and folding kinetics in various NA systems.