Efficient Conjugation and Characterization of Distamycin-Based Peptides with Selected Oligonucleotide Stretches

Selected sequences of oligodeoxyribonucleotides (ODNs) have been conjugated efficiently with distamycin-based peptides containing reactive cysteine and oxyamine functionalities at the C-terminus. The conjugation was performed easily within 30-60 min, using individual modified oligonucleotide stretches having sequences of 5ʹ-d(GCTTTTTTCG)-3ʹ, 5ʹ-d(GCTATATACG)-3ʹ, and 5ʹAGCGCGCGCA-3ʹ. Two types of linkages were used for making the covalent connection: (i) a fivemembered thiazolidine ring and (ii) an oxime. These distamycin-like polyamide-ODN conjugates were then converted to the corresponding DNA duplexes using complementary oligonucleotide sequences. To elucidate the binding specificity of the distamycin-oligonucleotide conjugates, UV-melting temperature measurements were performed. These studies indicated that the distamycin-ODN conjugate favored binding with the duplex with sequence 5ʹ-d(GCTTTTTTCG)-3ʹ rather than 5ʹ-d(GCTATATACG)-3ʹ. On the other hand, no stabilization of the duplex with sequence 5ʹ-d(AGCGCGCGCA)-3ʹ was observed. UV results also suggest that the thiazolidine and oxime linkages do not significantly influence the process of distamycin binding to the minor groove surface of the DNA duplex. The results obtained from duplex UV-melting studies were further corroborated by a temperature-dependent study of the circular dichroism spectra of the conjugates and a fluorescence displacement titration assay using Hoechst 33258 fluorophore as a competitive binder for the minor groove. All these studies reinforce the fact that the specific stabilization of A/T rich DNA-DNA duplexes by distamycin was preserved upon conjugation with oligonucleotide stretches.