Cationic phosphoramidate alpha-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis...

Area of Science:

• Biochemistry
• Molecular Biology
• Oligonucleotide Chemistry

Background:

• Antisense oligonucleotides (ON) are investigated for therapeutic applications.
• Improving ON affinity for target RNA is crucial for enhanced efficacy.
• Cationic modifications can reduce electrostatic repulsion, potentially increasing ON-RNA binding.

Purpose of the Study:

• To evaluate the duplex stabilizing effects of introducing cationic phosphoramidate internucleoside linkages into alpha-anomeric ON.
• To assess the binding affinity and antisense potential of these modified ON against RNA targets.

Main Methods:

• Synthesis of cationic alpha-oligonucleotides with phosphoramidate linkages.
• Analysis of duplex thermal stability with single-stranded DNA and RNA targets.
• Assessment of RNase H substrate activity.
• Evaluation of steric-blocking antisense activity in cell-free and cell-based assays using a Hepatitis C virus target sequence.

Main Results:

• Cationic alpha-ON exhibited high binding affinity to single-stranded DNA and RNA.
• Duplex stabilization increased proportionally with cationic modifications, showing high thermal stability in fully modified ON.
• Stabilization was notably enhanced at low ionic strength.
• The resulting duplexes were not substrates for RNase H, and a single mismatch significantly reduced binding affinity (Tm penalty).
• Effective inhibition of translation was observed in both cell-free and cell-based assays without the need for vectorization.

Conclusions:

• Cationic alpha-ON are effective steric-blocking antisense agents with high affinity and sequence specificity.
• These modifications significantly enhance duplex stability, particularly at low ionic strengths.
• The demonstrated efficacy in cell culture without vectorization highlights their therapeutic potential for antisense applications.