Studies on the unique RNA duplex destabilization by an azoniacyclophane ? NMR titrations with mono- and oligonucleotides of the RNA and DNA types

Abstract

The mechanism of the unique destabilization of a folded RNA by the azoniacyclophane CP66 has been investigated by complexation studies with mono- and short single-stranded oligonucleotides as models. For the mononucleotides, NMR titrations revealed only a slight difference of binding free energy ?G between RNA-type nucleotides compared to DNA analogues, with ??G values of up to 1.3 kJ/mol. The complexation-induced NMR shifts (CIS values) both in the ligand and in the cyclophane were also similar for deoxyribo- and ribonucleotides, suggesting similar inclusion geometries in both series. Similar observations had been made earlier on the CIS effects of denatured single-strand polymers. Titrations with dinucleotides showed a slight preference for ribose derivatives, with ?G = ?11.2 kJ/mol for ApA and ?13.2 kJ/mol for ApG, and ?9.9 kJ/mol for dApdA. With the dinucleotides and with selected tri- and tetranucleotides, the NMR shielding effects indicated stronger binding to adenine amongst the nucleobases, with a preference for the 5?-end position where available. In view of the small affinity decrease from ribose to deoxyribose derivatives, possible differences in the geometric disposition of the cyclophane towards the major groove of the duplexes were analysed with a simplified point charge model. The phosphates in the RNA groove indeed matched the cationic ammonium centres of the ligand less well than did those in the larger B-DNA groove. The conclusion from these results is that the destabilization of duplex RNA relative to that of duplex DNA is caused at least in part by the differences in interaction between CP66 and the duplex species and not the single-stranded species.