Oligodeoxynucleotides containing conformationally constrained abasic sites: a UV and fluorescence spectroscopic investigation on duplex stability and structure.

I Pompizi, A Häberli, C J Leumann

Index: Nucleic Acids Res. 28(14) , 2702-8, (2000)

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Abstract

The synthesis and incorporation into oligodeoxy-nucleotides of two novel, conformationally restricted abasic (AB) site analogs are described. The stability of oligonucleotide 18mer duplexes containing one such AB site opposite any of the four natural DNA bases was investigated by UV melting curve analysis and compared to that of duplexes containing a conformationally flexible propanediol unit 1 or a tetrahydrofuran unit 2 as an AB site analog. No major differences in the melting temperatures (DeltaT(m) 0-3 degrees C) between the different abasic duplexes were observed. All AB duplexes were found to have T(m)s that were lower by 9-15 degrees C relative to a fully matched 18mer control duplex, and by 4-10 degrees C relative to the corresponding 19mer duplexes in which the AB site is replaced by a mismatched nucleobase. Thus we conclude that the loss of stability of a duplex that is encountered by removal of a nucleobase from the stack cannot be compensated with conformational restriction of the AB site. From the van't Hoff transition enthalpies obtained from the melting curves, it appears that melting cooperativity is higher for the duplexes containing the conformationally rigid AB sites. Fluorescence quenching experiments with duplexes containing the fluorescent base 2-amino-purine (2AP) opposite the AB sites showed a weak tendency towards more efficient stacking of this base in duplexes containing the conformationally constrained AB sites. Thus, such AB sites may structurally stabilize the cavity formed by the removal of a base. Potential applications emerging from the properties of such conformationally constrained AB sites in DNA diagnostics are discussed.