Oligonucleotide—Minor Groove Binder 1:2 Conjugates: Side by Side Parallel Minor Groove Binder Motif in Stabilization of DNA Duplex

Abstract

Synthetic polycarboxamides consisting of N‐methylpyrrole (Py), N‐methylimidazole (Im), N‐methyl‐3‐hydroxypyrrole (Hp) and β‐alanine (β) show strong and sequence‐specific interaction with the DNA minor groove when they form hairpin structures with side‐by‐side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide‐linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5′‐CGTTTATTp‐3′/5′‐AATAAACG‐3′ melts at 20°C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46°C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60°C. It is even higher than in case of “classic” octapyrrole hairpin ligand (Tm = 58°C). Sequence‐specific character of stabilization by two conjugated ligands was demonstrated for G:C‐containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and β units according to established recognition rules (ΔTm = 20°C). The two‐strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site‐specific ligands recognizing single‐ and double‐stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.

Keywords:

• Oligonucleotides
• Minor groove binders
• Conjugates
• Duplex stability
• Thermal denaturation
• Sequence specificity

Acknowledgments

This work was supported by INSERM, European Community (grant INTAS 01‐0638), French Ministry of Foreign Affaires (grant EGIDE 04542ND) and by short‐term grants from Centre National de la Recherche Scientifique, France (“poste rouge” for A.N.S.) and Muséum National d'Histoire Naturelle, Paris, France (invited researcher position for V.A.R.). Authors are grateful to C. Caux and A. Blond for NMR spectra, Dr. J.P. Brouard et L. Dubost for mass spectra to Région Ile‐de‐France, the French Ministry of Research and Technology and CNRS for financial support in the acquisition of the NMR spectrometer.