Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• Antisense technology utilizes oligonucleotides to target specific nucleic acid sequences.
• Polylysine-oligonucleotide conjugates have shown promise for enhanced therapeutic effects.
• The impact of peptide charge on oligonucleotide binding and activity requires further investigation.

Purpose of the Study:

• To synthesize and characterize oligodeoxyribonucleotide-peptide conjugates with varying positive charges.
• To evaluate the binding affinity of these conjugates to complementary DNA and RNA targets.
• To assess the functional antisense activity of the modified oligonucleotides.

Main Methods:

• Chemical synthesis of a 12-mer oligodeoxyribonucleotide coupled to (delta-ornithine)n cysteine peptides.
• Melting temperature (Tm) analysis to determine binding affinity to DNA targets.
• RNase H cleavage assays to confirm hybridization with RNA targets.
• Cell-free translation arrest assays to measure antisense activity against mRNA.

Main Results:

• Conjugation of peptides to the oligodeoxyribonucleotide was confirmed.
• Melting temperature (Tm) increased with higher net positive charge on the conjugated peptide, indicating enhanced DNA binding.
• RNase H cleavage confirmed hybridization with RNA targets.
• Translation arrest assays demonstrated increased mRNA targeting with higher peptide charge.

Conclusions:

• Positively charged peptide conjugation significantly enhances the binding affinity of oligodeoxyribonucleotides to both DNA and RNA targets.
• The degree of enhancement correlates directly with the net positive charge of the peptide.
• These findings support the development of peptide-oligonucleotide conjugates as potent antisense agents.