DNA-protein biding often results in global changes in the DNA topology, such as bending or kinking. For DNA to bend, there needs to be adjustments in the structural units that define the duplex conformation. The overall DNA conformation is defined by many factors, one of which is the """"""""pucker"""""""" preference of the ribose ring. While the furanose ring of a simple nucleotide is in dynamic equilibrium between a South (S) sugar pucker (2'-endo, B DNA-like) and a North (N) sugar pucker (3'-endo, A DNA/RNA-like), upon incorporation into a DNA strand, the furanose ring adopts a preferred conformation. In a typical B-like DNA duplex, the base pairs involved in a topological adjustment such as a bend assume an altered, more A-like (N) sugar pucker. Prearrangement of the DNA duplex to more closely resemble the bound state (""""""""bent"""""""" conformation) may increase the binding affinity or decrease the disassociation energy from a protein of interest. As outlined in project Z01 BC 006174, the preparation of unique synthetic nucleotide analogues based on a bicyclo 3.1.0 hexane template system has been refined and the conformation of the monomers studied. This modified scaffold can lock the sugar pucker in either an N or S conformation depending on the relative position of the base on the 3.1.0 scaffold. Modified N- thymidine and N-adenine nucleotides were inserted into the Dickerson Drew dodecamer (5'-CGCGAATTCGCG-3'), a prototypical B-type DNA. Biophysical data obtained through circular dichroism, differential scanning calorimetry, and NMR have provided evidence for the effects that the modified sugar unit(s) had on the DNA structure. In the last annual report, we had stated that both NMR chemical shift assignments and comprehensive thermodynamic and CD data for the oligomers where the thymidines were replaced by a locked N analogue were complete. We have also analyzed the residual dipolar coupling (RDC) in the context of a new procedure to rapidly assess bending in an oligomer where a high resolution structure is already known. Our NMR studies at 800 MHz clarified our original data and were then used in the analysis of the bending of the three T-substituted oligonucleotides. We showed that bending of the duplex progressively increases with the number and position of the substituted residues. This technique has the potential to define DNA bending by comparing data of residues that are not affected by the substitution. This will dramatically shorten the analysis time for the resolution of global changes of substituted DNA oligomers. In addition, we have examined the corresponding adenine-substituted oligomers by CD and NMR spectroscopies. Initial data suggest that these oligomers actually are stabilized relative to the native dodecamer. This would be consistent with the idea that preorganization of the nucleotides into a B-like (2'-endo) conformation would more efficiently facilitate assembly of the duplex. Along with assistant professor Justin Wu at the Ohio State University, we are starting to fully characterize the global folds of the substituted oligomers through the analysis of a full list of RDCs. We are currently devising a new synthetic procedure to prepare the locked N and S building blocks with specific 13C labeling for enhanced sensitivity in the NMR experiments.
| Wu, Zhengrong; Maderia, Melissa; Barchi Jr, Joseph J et al. (2005) Changes in DNA bending induced by restricting nucleotide ring pucker studied by weak alignment NMR spectroscopy. Proc Natl Acad Sci U S A 102:24-8 |
| Maier, Martin A; Choi, Yongseok; Gaus, Hans et al. (2004) Synthesis and characterization of oligonucleotides containing conformationally constrained bicyclo[3.1.0]hexane pseudosugar analogs. Nucleic Acids Res 32:3642-50 |
