Triplex formation as monitored by EPR spectroscopy and molecular dynamics studies of spin -probe -labeled DNAs

Molecular modeling has proven to be a powerful tool for studying structure and dynamics of biologically important molecules. Since the advent of nitroxide based spin-probes the electron paramagnetic resonance (EPR) study of spin-labeled macromolecules has been able to provide insight into structural and dynamics properties of DNAs, proteins, and related systems. Spin labels have been extensively used to study the dynamics of oligonucleotides. An example of this is 5-membered ring nitroxide 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine, which has been previously used in our laboratory to monitor triplex formation. Because of the difficult synthetic steps involved in the synthesis of this particular probe a new spin labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (6sp-uridine) is introduced in the current study. This spin label, 6sp, is readily prepared, in half the number of steps required for the previous one, and yet behaves in a spectroscopically analogous manner to its counterpart. The 6sp has been used here to detect the formation of a triplex DNA and to examine the relative rigidity of triplex DNA as compared to double stranded DNA using circular dichroism and EPR spectroscopy. Their EPR spectra show larger changes in response to differences in the mobility of the oligonucleotides they are attached to.;Extending their use in application to DNAs we have conducted Molecular Dynamics (MD) studies on six different oligonucleotides (ONs) molecules using the suite of programs contained in AMBER 5.0 with the Cornell force field. Quantum mechanical calculations at B3LYP level with standard 6-31G* basis set using Gaussian98 were performed. Together with available crystallographic data for different types of nitroxide molecules (Barone et al., J. Am. Chem. Soc. 1998, 120, 7069--7078), new parameters for NO· and sp-hybridized carbon moieties have been developed for the Cornell force field. MD simulations on single-stranded (ss), double-stranded (ds) and triple-stranded or triplex (tx) spin-probe labeled DNAs along with unmodified analogues have been studied over the course of 1 ns. Structural and conformational properties of DNA molecules are described from the analysis of the trajectories. Dynamics of the spin-label was characterized by correlation time (tau c). Our results indicate slower nitroxide motion associated with tx-DNA rather than ds- and ss-DNAs. The presence of spin labels has a substantial effect on the conformation of ss DNA, while ds- and tx-DNA is not affected by the introduction of labels. We have also shown that the presence of the spin-label has small stabilizing effect on ds and tx DNAs