Effect of PEG-Induced Liquid-Liquid Phase Separation on DNA-Topotecan Interactions

Area of Science:

• Biophysics
• Materials Science
• Drug Delivery

Background:

• Liquid-liquid phase separation (LLPS) is crucial for cellular organization and function.
• Understanding LLPS effects on small molecules and biomacromolecules is vital for advanced applications.
• Polyethylene glycol (PEG) is a common inducer of LLPS in biomolecular systems.

Purpose of the Study:

• To investigate how PEG-induced LLPS affects the photophysical properties of topotecan (TPT).
• To examine the impact of PEG-induced LLPS on the structural organization of calf thymus DNA (ctDNA).
• To explore these effects in binary (PEG:TPT, PEG:ctDNA) and ternary (PEG:TPT:ctDNA) systems.

Main Methods:

• Spectroscopic techniques (fluorescence, absorption) to monitor photophysical changes.
• Microscopy to analyze condensate morphology.
• Molecular dynamics (MD) simulations to elucidate molecular interactions and structural changes.

Main Results:

• PEG-induced LLPS shifted TPT from its Z-form to the C-TPT form, enhancing fluorescence and lifetime, and suppressing excited-state proton transfer (ESPT).
• LLPS significantly altered ctDNA structure, quenching B-form signals and causing spectral blue shifts.
• Ternary systems showed similar TPT behavior, though DNA binding partially mitigated PEG's effects; MD simulations confirmed crowding and enhanced TPT-DNA interactions.

Conclusions:

• PEG-mediated LLPS can effectively tune the excited-state dynamics of small molecules like TPT.
• LLPS influences the structural organization of nucleic acids such as ctDNA.
• Findings provide insights into controlling molecular behavior in crowded environments for drug delivery and biomolecular engineering.