Phosphorothioate-phosphodiester oligonucleotide co-polymers: assessment for antisense application

Area of Science:

• Biochemistry
• Molecular Biology
• Oligonucleotide Chemistry

Background:

• Natural oligonucleotides (all-PO) have limitations for antisense applications.
• Phosphorothioate (PS) linkages can improve oligonucleotide properties.
• Copolymers of PO and PS offer a potential solution to balance stability and activity.

Purpose of the Study:

• To synthesize and evaluate oligodeoxynucleotide copolymers with varying PO and PS content.
• To assess protein-binding, nuclease stability, hybridization, RNase H sensitivity, and antisense activity.
• To determine the optimal PO/PS ratio for antisense applications.

Main Methods:

• Synthesis of 17-mer oligodeoxynucleotide copolymers with different PO/PS ratios.
• Melting temperature (Tm) analysis for hybridization ability.
• Protein-binding studies using human serum albumin.
• Nuclease stability assays in vitro.
• Antisense activity assessment in cell-free systems (WGA and RRL).
• RNase H cleavage assays.

Main Results:

• Copolymers exhibited intermediate hybridization abilities, with Tm reductions up to 6°C.
• Protein binding correlated linearly with PS content.
• Nuclease susceptibility improved, influenced by PS linkage number and position.
• Antisense activity (translation inhibition) was observed in WGA extract, dependent on RNase H.
• Copolymers with ≥50% PS linkages showed significant inhibition in WGA.

Conclusions:

• Oligodeoxynucleotide copolymers with mixed PO and PS linkages demonstrate enhanced properties for antisense applications.
• A balance of PO and PS linkages may be superior to all-PO or all-PS oligonucleotides.
• These copolymers represent a promising strategy for developing effective antisense therapies.