The crystal structure analysis of d(CGCGAASSCGCG)
2
, a synthetic DNA dodecamer duplex containing four 4
'
-thio-2
'-deoxythymidine nucleotides
The crystal structure analysis of d(CGCGAASSCGCG) 2 , a synthetic DNA dodecamer duplex containing four 4 ' -thio-2 '-deoxythymidine nucleotides
Titus J.
Boggon
,
E. Louise
Hancox
1
,
Katherine E.
McAuley-Hecht
2
,
Bernard A.
Connolly
3
,
William N.
Hunter
,
Tom
Brown
2,+
,
Richard T.
Walker
1
and
Gordon A.
Leonard*
Department of Chemistry, University of Manchester, Oxford Road,
Manchester
M13 9PL,
UK
,
1
School of Chemistry, University of Birmingham,
Birmingham
B15 2TT,
UK
,
2
Department of Chemistry, University of Edinburgh, King's Buildings, West Mains
Road,
Edinburgh
EH9 3JJ,
UK
and
3
Department of Biochemistry and Genetics, University of Newcastle-upon-Tyne,
Newcastle-upon-Tyne
NE2 4HH,
UK
Received September 19, 1995;
Revised and Accepted December 15, 1995
ABSTRACT
The crystal structure refinement of the synthetic dodecamer d(CGCGAASSCGCG),
where S = 4
'
-thio- 2
'
-deoxythymidine, has converged at
R
= 0.201 for 2605 reflections with
F
> 2
[sigma]
(
F
) in the resolution range 8.0-2.4 Å 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 (
1
-
5
), 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 sulphur-containing 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
Eco
RV 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
Eco
RV 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 Mg
2+
ion required for enzyme activity was not present in the structure.
MATERIALS AND METHODS
Chemical synthesis
The oligonucleotide was synthesized (10 [mu]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 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 [lambda] = 0.882 Å on station PX9.6 at the Synchrotron
Radiation Source, Daresbury Laboratory, to a resolution limit of 2.35 Å using a 18 cm MAR Research image plate detector and 17 4o 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 Å on a Rigaku RU200 rotating anode generator ([lambda] = 1.5418 Å) operating at 50 kV, 140 mA and equipped
with a RAXIS IIc image plate system. This latter data set was collected on 39 3o 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
R
sym
= 0.065 to a resolution of 2.4 Å and a multiplicity of 6.3.
Orthorhombic unit cell dimensions of a = 24.99 Å, b = 40.36 Å, c = 65.99 Å and space group P2
1
2
1
2
1
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 refinement, was carried out
using the program X-PLOR (
12
,
13
) and converged with
R
= 0.333 for the 2605 reflections with
F
> 2[sigma](
F
) in the resolution range 8.0-2.4 Å. The refinement then continued, using the same data, with a round of simulated
annealing-based minimization. This consisted of initial energy minimization of the
model followed by a slow cooling procedure (initial temperature 1000 K, final
temperature 300 K, temperature decrement 25 K, time step 0.5 fs) and one round
(120 cycles) of Powell minimization. Throughout this procedure and for
subsequent rounds of refinement the dictionary of restraints used was that
provided with X-PLOR and modified to include C-S bond length and C-S-C bond angle values for 4'-thio-2'-deoxy-thymidine,
which were taken from Dyson
et al.
(
3
). It should be noted that the values used for dihedral angles and improper
angles in the modified nucleoside were those already in place for 2'-deoxythymidine.
RESULTS AND DISCUSSION
In the structure of the modified dodecamer the nucleotides are labelled C1-G12 on strand 1 and C13-G24 on strand 2, both in the 5' -> 3' direction. The 66 solvent molecules are
labelled HOH25-HOH91.
As indicated by nucleotide sequence, unit cell parameters and space group, the structure of d(CGCGAASSCGCG) is isomorphous with that of the native dodecamer (
9
). The parameters that define global double helical conformation do not differ
significantly between the two structures. A least squares superposition of the
two double helices (Fig.
3
) shows the r.m.s. difference in atomic positions between the two structures to
be 0.49 Å and also suggests that the global conformation of the two structures is
extremely similar. However, a closer inspection of the two superposed
structures reveals that incorporation of the four 4'-thiosugar moieties into the sequence has led to some significant differences in the local structure of the two duplexes. This view
is supported by an examination of Figure
4
. Figure
4
a shows the difference in position for every atom in the two duplexes. As can be seen, on each of the two strands the deviation in atomic position remains
fairly constant until one reaches a point in the vicinity of the 4'-thio-2'-deoxythymidine residues, when the deviations
increase to a maximum before falling away to background level. Although the magnitudes of the positional differences are somewhat greater on
strand 1 than strand 2, in both cases the distribution is rather symmetrical,
which indicates that these deviations are real and not artefacts of the
refinement procedure. Figure
4
b shows that for an average deviation per nucleotide the differences in position
reach their peak for residues S8 and S20, which are the second of the two
modified nucleotides on strands 1 and 2 respectively.
The above analysis refers to the superposition of the modified dodecamer onto
the coordinate set 1BNA (
9
) as deposited in the PDB. There have been several other determinations of the structure of d(CGCGAATTCGCG), either at low temperature (deposition 2BNA in the PDB;
17
) or using different refinement techniques (depositions 7BNA, 9BNA in the PDB;
18
,
19
). The refined structure of d(CGCGAASSCGCG) has been superimposed on each of these
coordinate sets (Figure
4
a), yielding similar results to those found when the structure of
d(CGCGAASSCGCG) is superimposed in the original 1BNA coordinate set. This is further evidence
that the differences we observe between the structure of d(CGCGAASSCGCG) and
that of the native doecamer are real. Moreover, when the coordinate sets 2BNA, 7BNA and 9BNA are
superimposed on those of 1BNA (Fig.
4
c) there is no significant increase in the r.m.s. deviations of the sugar atoms
in nucleotides 7, 8, 19 and 20 when compared with the rest of the atoms,
although, inexplicably, for the 1BNA versus 9BNA superposition anomalously large deviations occur for the C6 and M5 atoms of nucleotides 7, 8, 19 and 20.
Incorporation of the 4'-thiosugars into the sequence does not greatly affect the positions
of the bases when both the modified and native structures are compared (Fig.
3
), but rather they induce a change in the conformation of the sugar-phosphate backbone in the vicinity of the modified sugars. Indeed, the
greatest deviations in the positions of the atoms in the modified and native
(1BNA) dodecamers occurs in the phosphodiester linkages on the 5'-side of the thiosugars themselves (Fig.
5
)
This observation, coupled with the fact that our analysis of the structure of
d(CGCGAASSCGCG) reveals that the major effect of incorporation of 4'-thio-2'-deoxyribose groups into a DNA duplex is to
change its backbone conformation, suggests the following reasons for the
resistance to nuclease digestion of oligonucleotides containing 4'-thiosugars. (i) Changes in the backbone conformation result in the
enzyme being able to bind less well to the modified DNA than to its unmodified
cognate sequence, thus causing a reduction in activity of the endonuclease;
(ii) changes in backbone conformation resulting from DNA modification cause a
reduction in Mg
2+
ion binding, which again would result in a reduction in the activity of the
enzyme. The decision as to which, if either, of these two hypotheses is correct
would require further analysis of the structures of 4'-thiosugar-modified DNAs, both in isolation and, more importantly,
complexed to their specific restriction endonucleases.
We conclude with the observation that the inclusion of 4'-deoxy-2'-thiothymidine into DNA has a small, but
detectable, effect on the backbone conformation of a DNA duplex. On occasions
this may appear to be of little significance, but should it be associated with
the conferring of increased nuclease resistance or increased lipophicity it may
make such oligodeoxynucleotides candidates for antisense molecules.
ACKNOWLEDGEMENTS
This work was supported by the United Kingdom Engineering and Physical Sciences
Research Council, the Biotechnology and Biology Science Research Council and the Wellcome Trust. We are grateful to the
Wellcome Foundation for financial support (to ELH). We thank Drs Miroslav Papiz
and Pierre Rizkallah of the Synchrotron Radiation Source, Daresbury Laboratory, for the allocation of
contingency beam time, part of which was used to collect the data described
here. TB wishes to thank the Royal Society of Edinburgh for a Caledonian
Research Fellowship.
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