The synthetic unmodified oligodeoxyribonucleotides (PO-oligos) forming higher order structures are believed to be alternative to chemically modified oligonucleotides in base, sugar and/or phosphate moiety in designing specific inhibitors of translation or/and the catalytic activity of many enzymes. It is well known that the cytosine-rich DNA sequences at acidic pH can adopt the cytosine tetraplex DNA structure (motif-i DNA) consisting of two partially protonated, parallel stranded duplexes. However, very limited information is available on the relationship between motif-i - forming oligonucleotides and their biological activity. In the present work we have examined the ability of homocytosine oligonucleotides of varying chain lengths (d[C₁₂], d[C₂₀], d[C₃₀], d[C₄₀]) to form the motif-i structure by circular dichroism (CD) and native gel electrophoresis experiments. The CD spectra obtained for d[C₃₀] and d[C₄₀] in PBS at 37°C demonstrate a strong positive band near 258 nm and smaller negative one around 260 nm with a cross over near 270 nm, These data provide evidence that homocitidine oligonucleotides d[C₃₀] and d[C₄₀] adopt the motif-i structure. In contrast, no folding of d[C₁₂] and d[C₂₀] into motif-i was detected under analogous conditions.

Further, we show that the motif-i structure formation is correlated with a significant inhibition of phosphodiesterase activity of plasma 3’-exonuclease of human recently identified as the soluble form of NPP1 (1-2). The half-life of d[C₃₀] and d[C₄₀] increases by factors of 6 and 12, respectively, compared with that of d[C₁₂]. The oligonucleotide d[C₃₀] was digested to a ladder of products from C₂₉ to C₂₈. In the case of d[C₄₀], the enzyme cleaved off only the first nucleotide from the 3’-end.

Our findings indicate that unmodified oligonucleotides forming motif-i at low concentrations under physiological conditions might be considered as potential inhibitors of the soluble NPP1, with less complications regarding toxicity effects often observed with PS-oligos.

1. M.Koziołkiewicz, M.Wójcik, A.Kobylańska, B.Karwowski, B.Rębowska, P.Guga, W.J. Stec, 1997, Antisense and Nucleic Acid Drug Development, 7, 43-48.

2. M.Wojcik, M.Cieslak, W.J.Stec, J.W.Goding, M.Koziolkiewicz, 2007, Oligonucleotides (in press).

This work was supported in part by Medical University of Łódź (project 502-17-692)

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