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 oligodeoxynucleotides 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 oligoeoxynucleotides 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'-thiothymidine), was neither a substrate for the endonuclease nor was recognized by the associated methylase. When still within the recognition hexanucleotide but two further residues removed from the site of cleavage (i.e. GATAXC), the oligodeoxynucleotide 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'-thionucleosides into synthetic oligodeoxynucleotides may shed light on subtle interactions between proteins and their normal substrates and may also show why 2'-deoxy-4'-thiothymidine itself is so toxic in cell culture.

Specific binding by EcoRV endonuclease to its DNA recognition site GATATC

“…H -S-phosphorothiolate (Vyle et al, 1992;Szczelkun & Connolly, 1995) and a second in which the deoxyriboses at the scissile bonds in both strands are replaced with 4 H -thiodeoxyribose (Hancox et al, 1993;Szczelkun & Connolly, 1995). The equilibrium association constants (K 0 ) in the absence of divalent cation (see the legend to Figure 6) do not differ signi®cantly for the parent 18 bp oligonucleotide (sequence 3, Table 1) and the phosphorothiolate-modi®ed 18-mer; K 0 is only slightly worse (3.1-fold) for the 4 H -thiodeoxyribosemodi®ed 18-mer.…”

“…Complementary single strands were annealed, and the products were con®rmed to be >99% in duplex form by non-denaturing gel electrophoresis (16% polyacrylamide, 89 mM Tris-borate, 2 mM EDTA, pH 8.0). Puri®ed oligomers containing a 3 H -S-phosphorothiolate linkage (Cosstick & Vyle, 1989;Vyle et al, 1992) and puri®ed oligomers containing a 4 H -thiothymidine residue (Hancox et al, 1993) were generously supplied by Dr B. A. Connolly.…”

Kinetic Analysis of a Mutational Hot Spot in the EcoRV Restriction Endonuclease

“…The requirement for precise positioning of the Q loop had been noted previously in studies with a DNA analog where the thymidine adjacent to the scissile bond had been replaced with 4′-thiothymidine, a derivative with sulfur in place of the oxygen in the furanose ring. Oligonucleotides containing this derivative are not cleaved by EcoRV (Hancox et al, 1993), and X-ray crystallography on EcoRV bound to such an oligonucleotide in the presence of MgCl 2 showed that the Mg 2+ ions seen at the active site of wild-type EcoRV were not present with this derivative (D. Kostrewa and F. Winkler, personal communication). However, the Q loop had been displaced as a result of the sulfur atom in the 4′-thiothymidine occupying part of the position for Asn70.…”

4′-Thio-5-Ethyl-2′-Deoxyuridine5′-Triphosphate (TEDUTP): Synthesis and Substrate Properties in DNA-Synthesizing Systems

“…Studies with chemically synthesized oligonucleotides containing 4'-thlo-z -deoxynucleotides have shown (Hancox et al, 1993;Boggon et al, 1996; GD. Jones and RT Walker, unpublished results), that 4'-thiothymidine destabilizes a DNA helix very slightly and TEDU causes substantial destabilization.…”