Nucleotides Bearing Red Viscosity-Sensitive Dimethoxy-Bodipy Fluorophore for Enzymatic Incorporation and DNA Labeling

Area of Science:

• Organic Chemistry
• Biochemistry
• Molecular Biology

Background:

• Development of fluorescent probes for biological applications is crucial.
• Viscosity-sensitive probes offer unique insights into cellular microenvironments.
• Nucleoside analogs can be functionalized to create novel biological tools.

Purpose of the Study:

• To design and synthesize novel fluorescent nucleosides and dNTPs.
• To evaluate their properties as viscosity sensors.
• To demonstrate their utility in DNA synthesis and live-cell imaging.

Main Methods:

• Chemical synthesis of modified nucleosides and dNTPs with a BODIPY fluorophore.
• Characterization of fluorescence properties and viscosity sensitivity.
• Enzymatic incorporation into DNA using DNA polymerases.
• In vitro and in vivo studies for DNA labeling and imaging.

Main Results:

• Synthesized dNTPs exhibited bright red fluorescence and viscosity sensitivity (lifetime change from 1.6 to 4.5 ns).
• Modified dNTPs served as substrates for DNA polymerases, enabling enzymatic DNA synthesis.
• Labeled DNA probes functioned as viscosity sensors, responding to protein binding.
• A longer-linker nucleotide was incorporated into genomic DNA in live cells.

Conclusions:

• Novel BODIPY-based nucleosides and dNTPs are effective fluorescent viscosity sensors.
• These probes can be used for enzymatic DNA labeling and real-time imaging of DNA synthesis.
• Cellular incorporation of modified nucleotides opens avenues for in vivo DNA staining and dynamic imaging.