Far-red switching DNA probes for live cell nanoscopy

Area of Science:

• Molecular Biology
• Biophysics
• Microscopy

Background:

• Accurate visualization of chromatin nanostructures is crucial for understanding cellular processes.
• Existing DNA probes often require washing steps or lack sufficient brightness for super-resolution imaging in living cells.

Purpose of the Study:

• To develop novel DNA probes for efficient and bright imaging of chromatin nanostructures.
• To enable wash-free super-resolution microscopy in living cells.

Main Methods:

• Synthesis of DNA probes combining Hoechst 33258 with a blinking far-red hydroxymethyl silicon-rhodamine (HMSiR) dye.
• Characterization of probe performance, including fluorescence enhancement upon DNA binding.
• Application of the best performing probe (5-HMSiR-Hoechst) in wash-free 3D stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM).

Main Results:

• A novel DNA probe, 5-HMSiR-Hoechst, was synthesized, featuring the 5'-regioisomer.
• The probe exhibits a significant ~400-fold fluorescence increase upon binding to DNA.
• The 5-HMSiR-Hoechst probe is compatible with wash-free SMLM and 3D STED microscopy.

Conclusions:

• The developed 5-HMSiR-Hoechst probe offers a bright and efficient tool for DNA visualization.
• This probe facilitates advanced super-resolution imaging of chromatin nanostructures in living cells without requiring washing steps.
• The findings open new avenues for studying dynamic chromatin organization at the nanoscale.