SYNTHESIS AND PROPERTIES OF OLIGODEOXYNUCLEOTIDES CONTAINING THE ANALOG 2'-DEOXY-4'-THIOTHYMIDINE

The 2'-deoxythymidine analogue 2'-deoxy-4'-thlothymidine has been inco rporated, using standard methodology, into a series of dodecadeoxynucl eotides containing the EcoRV restriction endonuclease recognition site (GATATC). The stability of these oligodeoxynucleotides and their abil ity to act as substrates for the restriction endonuclease and associat ed methylase have been compared with a normal unmodified oligodeoxynuc leotide. No problems were encountered in the synthesis despite the pre sence of a potentially oxidisable sulfur atom in the sugar ring. The a nalogue had very little effect on the melting temperature of the self- complementary oligoeoxynucleotides so synthesised and all had a CD spe ctrum compatible with a B-DNA structure. The oligodeoxynucleotide cont aining one analogue in each strand within the recognition site, adjace nt to the bond to be cleaved (i.e. GAXATC, where X is 2'-deoxy-4'-thio thymidine), was neither a substrate for the endonuclease nor was recog nized by the associated methylase. When still within the recognition h exanucleotide but two further residues removed from the site of cleava ge (i.e. GATAXC), the oligodeoxynucleotide was a poor substrate for bo th the endonuclease and methylase. Binding of the oligodeoxynucleotide to the endonuclease was unaffected but the k(cat) value was only 0.03 % of the value obtained for the parent oligodeoxynucleotide. These res ults show that the incorporation of 2'-deoxy-4'-thionucleosides into s ynthetic oligodeoxynucleotides may shed light on subtle interactions b etween proteins and their normal substrates and may also show why 2'-d eoxy-4'-thiothymidine itself is so toxic in cell culture.