Oligonucleotides with mixed α,β-anomeric backbone have been proposed recently for the recognition of random DNA sequence via new triplex motif (Doronina and Behr, Chem. Soc. Reviews 26, 63-71 (1997)). In the present work we examined α- and β- anomers of cytidine as possible candidates to recognize AT and TA base pairs of the double stranded DNA. The binding properties of β-oligo(dC) were studied on a series of synthetic oligodeoxynucleotides by UV absorbtion spectroscopy, measurements of bound EtBr fluorescence polarization, circular dichroism (CD) and non-denaturing gel electrophoresis. The UV thermal denaturation, polarization studies and CD experiments with three stranded oligonucleotide 5'-((dC^α) (dC^β))₅-L-(dAT)₅-L-(dAT)₅ (L = triethyleneglycol linker) and other oligonucleotide models showed that the formation of semiprotonated oligocytidilic complexes takes place at low temperatures and neutral pH, rather than folding of the clip into intramolecular triplex. The low-temperature transition was observed in denaturation profiles of any oligonucleotide containing β- or mixed α,β- cytidine stretches at the concentration of 1 µM. Self-association of α,β-oligo(dC) was additionally confirmed by the appearance of two CD bands (at 290 and 265 nm) characteristic of CC⁺ base pairs. Despite the effective ability of &alpha,β-oligo(dC) to form self-associates, we succeeded in targeting 30-bp AT containing random DNA duplex by a 30-nt α,β-oligocytidilate as evidenced by non-denaturing gel electrophoresis. A complete binding of the duplex was observed at a 5-fold excess of the third strand at 15°C. Along with the formation of the three-stranded complex, self-association of mixed backbone oligo(dC) strands occurred.