Fluorescence Light-Up of G4 DNA Structures Using Azlactone-Based Probes

Area of Science:

• Molecular Biology
• Biochemistry
• Chemical Biology

Background:

• G-rich sequences in DNA and RNA form G-quadruplex (G4) structures.
• G4 structures play crucial roles in various biological processes.
• Understanding G4 topology, location, and function is vital in cellular and cell-free environments.

Purpose of the Study:

• To develop novel small-molecule fluorescent probes for G4 structure detection.
• To investigate the sensing capabilities of azlactone-based probes (AZL1-3) for specific G4 topologies.
• To evaluate the cellular localization and potential applications of these probes.

Main Methods:

• Synthesis of three azlactone-based fluorescent probes (AZL1-3).
• Fluorescence spectroscopy to quantify probe binding and light-up response with G4 DNAs (c-MYC, c-KIT1, HRCC).
• Cellular assays using HeLa cells to assess probe localization and cytotoxicity.

Main Results:

• AZL1-3 probes exhibited significant fluorescence light-up (65-135-fold) for parallel G4 topologies of c-MYC, c-KIT1, and mitochondrial HRCC G4 DNA.
• The lead probe, AZL1, demonstrated a 2:1 binding stoichiometry with c-KIT1 G4 DNA, interacting with G-quartets.
• Limited cytotoxicity was observed, with probes showing cytoplasmic localization in HeLa cells, including colocalization with lipid droplets.

Conclusions:

• Azlactone-based probes are effective tools for sensing G-quadruplex structures in cell-free conditions.
• The developed probes show promise for potential bioimaging and diagnostic applications.
• Further engineering of these probes could enhance their utility in cellular G4 detection.