Sequence-Specific Conjugates of Oligo(2′-O-methylribonucleotides) and Hairpin Oligocarboxamide Minor-Groove Binders: Design, Synthesis, and Binding Studies with Double-Stranded DNA

• Published in: Chemistry & biodiversity 2005-07, Vol.2 (7), p.936-952
• Main Authors: Novopashina, Darya S., Sinyakov, Alexandre N., Ryabinin, Vladimir A., Venyaminova, Alya G., Halby, Ludovic, Sun, Jian-Sheng, Boutorine, Alexandre S.
• Format: Article
• Language: English
• Subjects: Base Sequence; DNA - chemistry; Human immunodeficiency virus; Imidazoles - chemistry; Nucleic Acid Conformation; Nucleic Acid Hybridization; Oligoribonucleotides - chemistry; Oligoribonucleotides - metabolism
• ISSN: 1612-1872; 1612-1880
• DOI: 10.1002/cbdv.200590071

New conjugates of triplex‐forming pyrimidine oligo(2′‐O‐methylribonucleotides) with one or two ‘head‐to‐head’ hairpin oligo(N‐methylpyrrole carboxamide) minor‐groove binders (MGBs) attached to the terminal phosphate of the oligonucleotides with a oligo(ethylene glycol) linker were synthesized. It was demonstrated that, under appropriate conditions, the conjugates form stable complexes with double‐stranded DNA (dsDNA) similarly to triplex‐forming oligo(deoxyribonucleotide) (TFO) conjugates containing 5‐methylated cytosines. Kinetic and thermodynamic parameters of the complex formation were evaluated by gel‐shift assay and thermal denaturation. Higher melting temperatures (Tm), faster complex formation, and lower dissociation constants (Kd) of the triple helices (6–7 nM) were observed for complexes of MGB‐oligo(2′‐O‐methylribonucleotide) conjugates with the target dsDNA compared to the nonconjugated individual components. Interaction of MGB moieties with the HIV proviral DNA fragment was indicated by UV/VIS absorption changes at 320 nm in the melting curves. The introduction of thymidine via a 3′,3′‐type ‘inverted’ phosphodiester linkage at the 3′‐end of oligo(2′‐O‐methylribonucleotide) conjugates (3′‐protection) had no strong influence on triplex formation, but slightly affected complex stability. At pH 6.0, when one or two hairpin MGBs were attached to the oligonucleotide, both triplex formation and minor‐groove binding played important roles in complex formation. When two ‘head‐to‐head’ oligo(N‐methylpyrrole) ligands were attached to the same terminal phosphate of the oligonucleotide or the linker, binding was observed at pH >7.5 and at high temperatures (up to 74°). However, under these conditions, binding was retained only by the MGB part of the conjugate.