Inability of RNA to form the i-motif: implications for triplex formation.

At slightly acidic or even neutral pH, oligodeoxyribonucleotides which include stretches of cytidines form a tetrameric structure involving C.C+ base pairs in a so-called i-motif. Such a structure, which is very stable at pH 6, is still detectable at neutral pH. This motif, whether intramolecular or intermolecular, was shown to act as an undesired, competing structure for triplex formation. Depending on the sequence and the experimental conditions, triple helix formation was inhibited or completely abolished. Thus, the sequence recognition repertoire of pyrimidine DNA third strands is more restricted than previously admitted; only T.A rich-oligopurine-oligopyrimidine sequences are amenable to triplex formation. On the other hand, cytosine-rich RNA oligoribonucleotides and their 2'-O-methyl derivatives were unable to form a stable autoassociated structure above pH 6. Concomitantly, they were shown to form a triplex with (C.G)-rich targets at pH 6. Thus, not only was the affinity for the duplex increased by DNA to RNA substitution, but the possibility of formation of an inactive form of the third strand was greatly reduced. Thus, i-motif formation is favored for cytosine-rich oligodeoxynucleotides, whereas triplex formation is favored for oligoribonucleotides. These properties make RNA pyrimidine oligonucleotides an attractive choice for triplex formation on a (C.G)-rich target, extending the practical sequence repertoire of pyrimidine triplexes to (C.G*C+)-rich sequences.