POT1 stability and binding measured by fluorescence thermal shift assays.

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• The Protection of Telomeres 1 (POT1) protein is a key component of the shelterin complex, crucial for maintaining human chromosome end stability.
• POT1 uniquely binds to single-stranded DNA, playing a vital role in preventing chromosomal degradation and end-to-end fusions.

Purpose of the Study:

• To develop and apply quantitative biophysical assays for characterizing POT1 stability and its interactions.
• To elucidate the thermodynamic properties of POT1 folding and DNA binding.
• To identify mechanisms inhibiting POT1-DNA interactions.

Main Methods:

• Application of two distinct fluorescent thermal shift assays (FTSA).
• Sypro Orange™ FTSA for monitoring POT1 thermal stability and binding characteristics.
• FRET-labeled G-quadruplex FTSA for assessing POT1 effects from a DNA perspective.

Main Results:

• Quantitative data on POT1 folding thermodynamics and DNA binding sequence selectivity were obtained.
• The assays provided a detailed thermodynamic profile of POT1 binding to its preferred DNA sequence.
• Two distinct inhibition mechanisms of POT1-DNA interactions were identified: competitive binding and G-quadruplex stabilization.

Conclusions:

• Fluorescent thermal shift assays offer efficient and quantitative biophysical characterization of POT1.
• Understanding POT1 interactions and inhibition mechanisms is critical for telomere biology and potential therapeutic strategies.
• The developed assays can serve as quality control tools and screening platforms for drug discovery targeting POT1.