Genetically encoded FRET sensor for fructosyl valine detection in living cells

Area of Science:

• Biomedical Engineering
• Molecular Biology
• Analytical Chemistry

Background:

• Real-time monitoring of glycation dynamics in living cells is essential for diabetes mellitus management but currently lacks adequate tools.
• Fructosyl valine (FV), a direct indicator of HbA1c glycation, presents a viable target for developing novel detection strategies.

Purpose of the Study:

• To develop and characterize a genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensor, Fructosyl Valine Sensor (FruValSen), for quantifying FV in living cells.

Main Methods:

• Constructed FruValSen using the fructosyl amino acid binding protein SocA framed by ECFP and Venus fluorescent proteins.
• Assessed sensor stability, specificity, binding affinity (Kd), and dynamic range across various conditions and cell types (bacterial, yeast, mammalian).
• Utilized confocal microscopy, ratiometric imaging, and time-dependent FRET analysis for intracellular validation and dynamic monitoring.

Main Results:

• FruValSen demonstrated stability across pH variations and high specificity for FV, with no cross-reactivity to other molecules.
• The wild-type sensor exhibited a high binding affinity (Kd = 1.6 nM) and a broad dynamic range (10 pM to 300 μM), detecting FV at concentrations as low as 10 pM.
• Successful expression and cytosolic localization were confirmed in yeast, with real-time FRET analysis showing efficient intracellular FV binding and dynamic response in living cells.

Conclusions:

• FruValSen is a sensitive, specific, and real-time biosensor for non-invasive FV detection in living cells.
• This platform enables dynamic monitoring of FV at the single-cell level across diverse biological systems.
• The FruValSen biosensor holds significant potential for advancing diabetes research by providing new insights into glycation dynamics.