NMR studies of disulfide cross-linked DNA.

Abstract

Because DNA structural motifs exist in vivo as part of DNA molecules that are too large for biophysical characterization, it is necessary to incorporate the motifs into smaller constructs to carry out biophysical measurements. Our laboratory has developed a technique to stabilize small DNA constructs by introducing disulfide cross-links site-specifically. This thesis presents the results of NMR studies describing the structural and dynamic consequences of introducing these cross-links into DNA. A number of interesting findings made possible by the disulfide stabilization of DNA constructs are also presented. Structural studies of a 21-base-long DNA hairpin and a disulfide cross-linked analog reveal that the cross-link does not perturb the structure of the hairpin. The base-pair lifetimes and ratios of apparent dissociation constants for corresponding base-pairs in the two hairpins were determined by exchange rate studies. The cross-link does not alter the base-pairs opening dynamics of base-pairs that are not frayed. Additionally the small cross-link is more effective at stabilizing the stem against fraying than is the larger five-base loop. A cross-link introduced into an intramolecular triplex is shown to stabilize the triplex to increased pH and temperature. The cross-linked construct exists as a structurally homogeneous triplex under physiologically relevant conditions (e.g., pH 7.4, 155 mM Na$\sp+$, 37$\sp\circ$C), unlike the uncross-linked triplex which is predominately in the duplex conformation under these conditions. The cross-link allowed the first measurement ever of Hoogsteen and Watson-Crick base-pair lifetimes within a triplex. Both types of base-pairs open and close within a stable triplex. The Watson-Crick base-pair lifetimes are extended relative to a duplex. A novel protocol was devised that allows NOESY cross-peak intensities to be corrected for the use of a short recycle delay. Evidence is presented that this protocol will also be useful in protein structural determination when the magnetic fields and the size of proteins being studies are increased. It appears that stabilizing oligonucleotides by incorporating the cross-links presented here does not lead to structural perturbations and results in increased stability. Thus disulfide cross-linked oligomers such as those presented here should prove generally useful in studies of DNA thermodynamics, structure, and function.