SUNY at Albany
June 19-23, 2001
Investigation of Stacking and Hydrogen Bonding in DNA using NMR.
The structure and dynamics of Watson-Crick base pairs in RNA and DNA have been investigated using high resolution NMR spectroscopy. It will be shown that CH-dipole,dipole, N-CSA cross-correlated relaxation (G(HC,N)) is a sensitive measure for stacking interaction in RNA. The bond geometry of the correlated atoms is fixed and variations of G(HC,N) will probe size and orientation of the respective nitrogen CSA-tensor. Relaxation rates have been determined for the 25nt tau exon 10 splicing regulatory element RNA and the 37nt E.Coli 5SE RNA. The relaxation rates and the orientation of the nitrogen CS-tensors are found to be dependent on base stacking interactions more than on hydrogen-bonding directly. The rates found for nucleobases in Watson-Crick base paired stem regions are in agreement with experimental nitrogen CSA-tensor principal axis values (1)and calculated tensor orientation (2-4) In contrast, the rates for nucleobases that are not Watson-Crick base paired substantially deviate from these predictions.
A second topology of Watson-Crick base pairs in standard B-form DNA has been determined from analysis of the field dependence of 1H line width of imino hydrogen atoms. This second topology is distinct from the usual hydrogen bonded form but also distinct from the topology that is used to rationalize the exchange of imino and amino hydrogen atoms with water. Based on the analysis of chemical shifts of the imino hydrogen atoms in the new topology it is assigned to be a conformation in which hydrogen bonds are dislocated by less than 5pm. Such small dislocation will preserve the stacking interaction between neighboring nucleobases. Populations of the base pair open form that lacks both stacking and hydrogen bonds and the new intermediate form have been determined to be 10-5 and 10-2, respectively. These populations form a basis to dissect the contribution of stacking and hydrogen bonding and their variation along the oligonucleotide to the stability of Watson-Crick base pairs on the assumption that stacking interactions are preserved in the intermediate conformation.References and Footnotes
Elke Duchardt, Christian Richter, Christian von der Heyden, Christian Griesinger, and Harald Schwalbe*
Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, 170, Albany Street, Cambridge, MA 02138, USA.