The three-dimensional structure of DNA is quite dependent on sequence. This should be intuitively obvious from the sequence specificity required by many proteins for recognition. The few available x-ray crystal structures and NMR solution structures attest to this structural flexibility as well. The location and orientation of potential ligand binding functions on the DNA such as charges, hydrogen bonding and hydrophobic sites can be modified substantially from that which one might expect on the basis of assuming a canonical B-DNA structure. Consequently, we intend to continue development of the methodology for determination of high-resolution nucleic acid structures in solution and apply this methodology to some oligonucleotides and oligonucleotide complexes. This entails methods designed to improve the accuracy and resolution of the structures determined. Improved structures can be obtained with more accurate and more numerous experimental distance and torsion angle constraints, as well as improvements in calculating structure from these constraints. Enhancements will result from improvements in our iterative complete relaxation matrix program MARDIGRAS, development of a more encompassing density matrix approach for analysis of spectra derived from any pulse sequences (even those not yet invented), development of tailored excitation pulses, inclusion of experimental molecular motion information, and development of alternative methods of reducing experimental structural constraint data to structures. The latter includes (a) for restrained molecular dynamics simulations, use of improved force fields, empirical development of improved force fields, and use of constraint terms permitting a more realistic picture of conformational flexibility, and (b) development of an alternative restrained Monte Carlo method in torsion angle and helical parameter space, which is quite promising especially for structure refinement directly against NOE intensities. Applications will include oligonucleotides of interest, in particular sequences recognized by transcription factors or regulators, genome targets, antisense oligonucleotides, and a DNA microcircle duplex. Structures of proteins (including nucleic acid complexes) which are important for initiation or regulation of transcription will be determined. In particular, the 72-residue protein GerE which is a regulatory protein that binds specifically to a target site in promoter DNA will be the subject of study. Other proteins will be evaluated as possible candidates for study.
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