NMR Group Project: Biophysical Studies of Oligonucleotid

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DNA-protein binding 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 a 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. We have fully assigned the NMR chemical shifts of the oligomers where either one or both thymidine base pairs were replaced by a locked N-thymidine. Both CD and calorimetric data indicate that stable duplexes are formed in all 3 oligomers and the melt temperatures of the modified DNA's are quite similar to the native duplex. However, there are perturbations close to the mutation site upon insertion of a "N" nucleoside in an otherwise "S" DNA duplex. These perturbations are highly localized and do not transmit along the DNA chain as has been reported for other conformationally-biased DNA constructs. In order to determine the degree of bending imparted by the modified base pairs, we are collaborating with Dr. Ad Bax and have performed NMR studies at 800 MHz which allowed us to modify our original assignments made from experiments at lower field. We have calculated residual dipolar coupling (RDC) constants from natural abundance 13C-1H data on the three oligomers and have compared these to those measured for the native dodecamer by the Bax group. We are currently calculating the structures of the modified DNA's based on the RDC measurements.