A Click Chemistry Approach to Developing Molecularly Targeted DNA Scissors

Area of Science:

• Medicinal Chemistry
• Molecular Biology
• Biotechnology

Background:

• Gene-targeted technologies are crucial for novel therapeutic strategies.
• Oligonucleotide-based therapeutics require enhanced stability and targeting specificity.
• Click chemistry offers a versatile platform for conjugating functional ligands to oligonucleotides.

Purpose of the Study:

• To develop chemically modified triplex forming oligonucleotides (TFOs) using click chemistry for gene targeting.
• To enhance the stability and DNA-cleavage capabilities of TFOs through ligand conjugation.
• To create a new class of gene-targeted agents with improved sequence selectivity.

Main Methods:

• Nucleic acid click chemistry was employed to conjugate azide-bearing phenanthrene ligands to alkyne-modified parallel TFOs.
• A library of TFO-ligand hybrids was synthesized and characterized.
• In vitro studies were performed to assess triplex stability, DNA targeting, and copper-mediated DNA cleavage.

Main Results:

• The synthesized TFO hybrids effectively targeted purine-rich genetic elements in vitro.
• Several hybrids demonstrated significant stabilization of parallel triplexes, increasing melting temperatures by over 20°C.
• Copper binding, in the presence of a reductant, triggered DNA damage, showcasing synergistic TFO-ligand activity.
• A click chemistry-based di-copper binding ligand was developed, further improving targeted oxidative cleavage when incorporated into TFOs.

Conclusions:

• Chemically modified TFOs prepared via click chemistry represent a promising gene-targeting technology.
• The developed TFO hybrids offer enhanced stability and sequence-selective DNA damage capabilities.
• This approach provides a versatile platform for designing novel nucleic acid-based therapeutic agents.