R. C. Denny

A high-angle neutron fibre diffraction study of the hydration of deuterated A-DNA

A high-angle neutron fibre diffraction study of the hydration of A-DNA has been performed using the single-crystal diffractometer D19 at the Institut Laue-Langevin (Grenoble, France). The sample was prepared using deuterated DNA extracted from E. Coli cells cultured on deuterated nutrients. In common with our previous neutron fibre diffraction studies of DNA, this work exploits the ability to isotopically replace H2O around the DNA by D2O. However this study benefitted additionally from the fact that the hydrogen atoms which are covalently bonded to carbon atoms in the DNA sugars and bases were replaced by deuterium so that incoherent scattering and absorption effects were minimised. Successive cycles of Fourier synthesis and Fourier difference synthesis allowed water peaks to be identified and their positional and occupancy parameters to be refined against the observed diffraction data. The results confirm the main hydration features noted in our earlier studies with a clear network of water running along the inside edge of the major groove linking successive OI phosphate oxygen atoms. The central core of water running along the axis of the double helix is very much clearer in this work. Additionally this study shows chains of ordered water lying in the centre of the major groove.

Collecting and Processing Neutron Fibre Diffraction Data from a Single-Crystal Diffractometer

The geometries for scanning reciprocal space for a fibre on a neutron single-crystal diffractometer are considered. Geometrical relationships are derived for the particular case of D19 at the Institut Laue-Langevin that can be readily generalized for other instruments and for the same instrument with a different detection system. An optimum data-collection strategy is proposed. Methods for processing neutron fibre data collected using this strategy are described and illustrated by studies on cellulose.

Structural polymorphism in a tubercidin analogue of the DNA double helix

A high-angle X-ray fibre diffraction study of a tubercidin analogue of the poly[d(A-T)].poly[d(A-T)] DNA double helix has been carried out using station 7.2 at the Daresbury Laboratory synchrotron radiation source. The polymer has been studied for a wide range of salt strengths and hydration conditions and exhibits conformational polymorphism that is quite distinct from that observed for the unmodified polymer. The replacement of deoxyadenosine by deoxytubercidin in the polynucleotide causes only slight alterations to the structure of A-DNA, but significantly alters the structure of the B conformation. Additionally, the modified polymer does not, in any conditions yet identified, adopt the D conformation. In conditions which would normally favour the D conformation of poly[d(A-T)].poly[d(A-T)], the modified polymer adopts an unusual conformation which is designated here as the K conformation. These observations are important for an understanding of major groove interactions involved in the stabilisation of particular DNA conformations and also more generally for an insight into the pharmacological activity of tubercidin which following its incorporation into nucleic acids may cause stereochemical distortions of the DNA double helix.

A high-angle neutron fibre diffraction study of the hydration of B-DNA

A high-angle neutron fibre diffraction study of hydration in the B conformation of DNA has been performed on instrument D19 at the Institut Laue-Langevin, Grenoble, using a sample prepared by the ‘wet spinning’ technique. In common with our previous neutron fibre diffraction studies, this work exploited the ability to isotopically replace H2O in the specimen by D2O. However, in contrast to previous studies, the sample exhibited ‘double orientation’ so that it was possible to record a three-dimensional diffraction data set. Fourier synthesis techniques were used to image ordered water surrounding the DNA. Two chains of ordered water were identified in the minor groove of the molecule in positions that are consistent with water bridging of sugar O4 and purine N3 or pyrimidine O2 atoms.

Ordered water around deuterated A-DNA by neutron fibre diffraction

Abstract A neutron fibre diffraction study of A-DNA hydration has been carried out using instrument D19 at the Institut Laue-Langevin, Grenoble. In contrast to earlier experiments, deuterated DNA was used in order to minimise incoherent scattering and sample absorption effects. Fourier analysis of data collected from the sample in a D 2 O environment has revealed four distinct major groove ordered water sites. A network of ordered water involved in the bridging of successive O1 phosphate oxygen atoms along the double-helix backbone has been identified. Extensive hydration of the major groove base edges has been observed as a continuous core of density running down the helix axis. The other two hydration features are located in the centre of the major groove, one at the opening of the groove and the other located deep in the groove.