Synthesis and properties of oligodeoxynucleotides containing the analogue 2′-deoxy-4′-thiothemidine

Abstract

The 2′-deoxythymidine analogue 2′-deoxy-4′-thiothymidine has been incorporated, using standard methodology, into a series of dodecadeoxynucleotides containing the EcoRV restriction endonuclease recognition site (GATATC). The stability of these oligo-deoxynucleotides and their ability to act as substrates for the restriction endonuclease and associated methylase have been compared with a normal unmodified oligodeoxynucleotide. No problems were encountered in the synthesis despite the presence of a potentially oxidisable sulfur atom in the sugar ring. The analogue had very little effect on the melting temperature of the self-complementary oilgoeoxynucleotides so synthesised and all had a CD spectrum compatible with a B-DNA structure. The oligodeoxynucleotide containing one analogue in each strand within the recognition site, adjacent to the bond to be cleaved (i.e. GAXATC, where X is 2′-deoxy-4′-thlo-thymidine), was neither a substrate for the endonuclease nor was recognized by the associated methylase. When still within the recognition hexa nucleotide but two further residues removed from the site of cleavage (i.e. GATAXC), the oligodeoxynucieo tide was a poor substrate for both the endonuclease and methylase. Binding of the oligodeoxynucleotide to the endonuclease was unaffected but the Kcat value was only 0.03% of the value obtained for the parent oligodeoxynucleotide. These results show that the incorporation of 2′-deoxy-4′-thlonucleosides into synthetic oligodeoxynucleotides may shed light on subtle interactions between proteins and their normal substrates and may also show why 2′-deoxy-4′-thio-thymidine itself is so toxic in cell culture. © 1993 Oxford University Press.