The Crystal Structure Analysis of d(CGCGAASSCGCG)2, a Synthetic DNA Dodecamer Duplex Containing Four 4′-Thio-2′-Deoxythymidine Nucleotides

Titus J. Boggon 1 3 4 6 E. Louise Hancox 1 3 4 6 Katherine E. McAuley-Hecht 1 2 3 4 6 Bernard A. Connolly 0 1 3 4 6 William N. Hunter 1 3 4 6 Tom Brown 1 2 3 4 5 6 Richard T. Walker 1 3 4 6 Gordon A. Leonard 1 3 4 6 0 Department of Biochemistry 1 Edinburgh , King's Buildings, West Mains Road, Edinburgh EH9 3JJ, UK 2 Department of Chemistry, University of 3 Chemistry, University of Birmingham , Birmingham B15 2TT, UK 4 Department of Chemistry, University of Manchester , Oxford Road, Manchester M13 9PL, UK 5 Present address: Department of Chemistry, University of Southampton , Highfield, Southampton SO19 5NH, UK 6 Genetics, University of Newcastle-upon-Tyne , Newcastle-upon-Tyne NE2 4HH, UK The crystal structure refinement of the synthetic dodecamer d(CGCGAASSCGCG), where S = 4-thio2-deoxythymidine, has converged atR = 0.201 for 2605 reflections with F > 2s (F) in the resolution range 8.0-2.4 for a model consisting of the dodecamer duplex and 66 water molecules. A comparison of its structure with that of the native dodecamer d(CGCGAATTCGCG) has revealed that the major differences between the two structures is a change in the conformation of the sugar-phosphate backbone in the regions at and adjacent to the positions of the modified nucleosides. Examination of the fine structural parameters for each of the structures reveals that the thiosugars adopt a C3-exo conformation in d(CGCGAASSCGCG), rather than the approximate C1-exo conformation found for the analogous sugars in the structure of d(CGCGAATTCGCG). The observed differences in structure between the two duplexes may help to explain the enhanced resistance to nuclease digestion of synthetic oligonucleotides containing 4-thio-2-deoxynucleotides. - INTRODUCTION The biological properties of 4-thionucleosides (15), which are sulphur-containing isoelectronic analogues of natural nucleosides (Fig. 1), are not always apparent from a comparison with those of the corresponding oxysugar-containing deoxynucleoside. Hence, while (E)-5-(2-bromovinyl)-2-deoxyuridine and its sulphurcontaining analogue both show potent and selective inhibition of HSV-1 and varicella zoster virus (VZV), the strong antiviral properties of 5-isopropyl- and 5-cyclopropyl-4-thio-2-deoxyuridine are markedly different to those of the corresponding natural nucleosides, which show no antiviral activity at all. Similarly, 5-vinyl-2-deoxyuridine is very toxic, whereas the corresponding 4-thio analogue shows no toxicity, but is active against HSV-1. Some of the most interesting properties of pyrimidine 4-thio-2-deoxynucleotides are that they are significantly more lipophilic than oxysugar-containing deoxynucleotides and have a much longer serum half-life than normal nucleotides, the latter property arising because they are stable to phosphorolysis. This led to the suggestion that oligonucleotides containing pyrimidine 4-thio-2-deoxynucleosides might be useful in the antisense field, especially as structure determination had shown the modified nucleosides to adopt similar conformations to those of the natural nucleosides (6). Additionally, circular dichroism and ultraviolet melting studies (7) of thiosugar nucleoside-containing oligonucleotides had shown them to adopt the same overall conformation as the analogous unmodified oligonucleotides and that the duplexes were only slightly less thermodynamically stable. Experiments involving the synthesis of oligonucleotides containing the EcoRV recognition site revealed that when a 4-thiothymidine residue was incorporated on the 5-side of the scissile bond the DNA duplex was resistant to endonuclease attack (7). The reasons for this were not at all clear, as a crystal structure of the complex between EcoRV and the modified duplex (Kostrewa,D., Hancox,E.L., Walker,R.T., Connolly,B.A. and Winkler,F.K., unpublished results; 8) showed that the conformation of the DNA had not significantly changed. However, The Mg2+ ion required for enzyme activity was not present in the structure. As part of a continuing investigation into the structures and biological properties of oligonucleotides containing 4-thio-2deoxynucleosides we report here the results of an X-ray structural analysis of the self-complementary dodecamer d(CGCGAASSCGCG), where S = 4-thio-2-deoxythymidine (Fig. 1). The observed * To whom correspondence should be addressed structure is compared with that of the native dodecamer d(CGCGATTCGCG) (9) and the structural reasons for the resistance of 4-thiosugar-containing oligonucleotides to endonuclease attack are explored. MATERIALS AND METHODS Chemical synthesis The oligonucleotide was synthesized (10 m M scale) using an Applied Biosystems 381A DNA synthesizer. The modified deoxynucleoside phosphoramidite (7) was introduced at the fifth position of the synthesis as previously described (7). The synthesis and purification of the oligodeoxynucleotide was achieved under standard conditions. Crystallization, X-ray data collection and structure refinement Thin, colourless, needle-shaped crystals were grown at ambient temperature in sitting drops which contained the dodecanucleotide (0.5 mM), sodium cacodylate buffer (14 mM, pH 6.0), magnesium chloride (17 mM), spermine tetrahydrochloride (1 mM) and NNuucclleeiicc AAcciiddssRReesseeaarrcchh,,11999946,,VVool.l.2224,,NNoo..15 2-methyl-2,4-pentanediol (MPD) (17% v/v), which were diffused against external reservoirs containing 50% aqueous MPD. Two such crystals were then mounted in thin-walled glass capillaries for X-ray data collection. Data from the first of these, which had dimensions of 1.0 0.05 0.05 mm, was collected at l = 0.882 on station PX9.6 at the Synchrotron Radiation Source, Daresbury Laboratory, to a resolution limit of 2.35 using a 18 cm MAR Research image plate detector and 17 4 oscillation frames, each exposed for 120 s. This data was later supplemented by a set collected from a bigger crystal (1.5 0.15 0.15 mm) to a resolution limit of 2.5 on a Rigaku RU200 rotating anode generator (l = 1.5418 ) operating at 50 kV, 140 mA and equipped with a RAXIS IIc image plate system. This latter data set was collected on 39 3 oscillation frames, each of which were exposed to X-rays for 1 h. All the data was processed using the program MOSFLM (version 5.20) (10), then scaled, merged and reduced using programs from the CCP4 suite of programs (11) to yield a total of 2820 unique reflections with Rsym = 0.065 to a resolution of 2.4 and a multiplicity of 6.3. Orthorhombic unit cell dimensions of a = 24.99 , b = 40.36 , c = 65.99 and space group P212121 suggested that the structure of d(CGCGAASSCGCG) is isomorphous with that of the native dodecamer d(CGCGAATTCGCG) (9). The initial stage of the structure refinement therefore consisted of rigid body minimization, against the observed data, of the structure of the native dodecamer stripped of solvent molecules and with sulphur atoms replacing oxygen atoms where necessary. This procedure, as with the rest of the ref (...truncated)