pH-independent triple-helix formation with 6-oxocytidine as cytidine analogue

The syntheses of six different phosphoramidite building blocks of 6-oxocyto pine and 5-allyl-6-oxocytosine as analogues of N(3)-protonated cytosine are described. These compounds have been incorporated into oligonucleotides by standard solid-phase synthesis Hybridization of 15-mer Hoogsteen strands w ith target 21-mer duplexes was investigated. Comparison of the triples-form ing abilities of the different building blocks revealed that: i) 5-allyl su bstitution has a negative influence on tripler stability: ii) a uniform bac kbone of the Hoogsteen strand stabilizes triplexes relative to mixed backbo nes; iii) RNA strands with 6-oxocytidine or 5-allyl-6-oxocytidine do not fo rm a triple helix with the DNA target duplex, probably due to backbone tors ional constraints; and (iv) a 15-mer DNA sequence with three isolated 2'-de oxy-6-oxocytidines has the highest T-m of all cytidine analogues investigat ed in this study. CD experiments provided further evidence for the presence or absence of tripler structures. In the course of these temperature-depen dent CD measurements we were able to detect duplex and tripler melting inde pendent from each other at selected wavelengths. This methodology is especi ally interesting in cases where UV melting curves show only one transition owing to spectral overlap.