Anti-gene oligonucleotide clamps invade dsDNA and downregulate huntingtin expression

Area of Science:

• Molecular biology
• Medicinal chemistry
• Genetics

Background:

• Anti-gene oligonucleotides target DNA, distinct from antisense oligonucleotides that target RNA.
• Clamp anti-gene oligonucleotides utilize a double-stranded DNA invasion mechanism.

Purpose of the Study:

• To investigate the design of locked nucleic acid-DNA mixmer clamp anti-gene oligonucleotides for targeting specific gene sequences.
• To evaluate the role of an intercalating moiety (M3) in enhancing DNA strand invasion.
• To assess the in vivo efficacy and therapeutic potential of these modified oligonucleotides.

Main Methods:

• Design and synthesis of 30 locked nucleic acid-DNA mixmer clamp anti-gene oligonucleotides with and without an M3 moiety.
• Assessment of strand invasion efficiency in relation to M3 linker position and DNA sequence composition (GC-rich regions).
• Evaluation of phosphorothioate-modified clamps for in vivo applications and their binding kinetics compared to phosphodiester clamps.
• Demonstration of mRNA down-regulation in patient-derived fibroblasts using a Huntingtin gene-targeting clamp oligonucleotide.

Main Results:

• The presence of the M3 moiety as a linker was crucial for effective strand invasion.
• Optimal linker placement adjacent to GC-rich sequences enhanced DNA invasion.
• Phosphorothioate modification resulted in slower binding kinetics but achieved comparable strand invasion to phosphodiester clamps.
• A single-site targeting clamp oligonucleotide significantly reduced Huntingtin gene mRNA levels in patient fibroblasts.

Conclusions:

• Modified clamp anti-gene oligonucleotides with intercalating moieties are effective DNA-targeting agents.
• Design strategies involving linker placement and base composition significantly influence invasion efficiency.
• Phosphorothioate modification is suitable for in vivo applications, despite altered kinetics.
• These findings support the potential of anti-gene oligonucleotide therapeutics for gene regulation and disease treatment.