Investigation of oligonucleotide structure by NMR methods

Kaluarachchi Kankanamge Indrasiri Kumaralal, Purdue University

Abstract

Instrumental and methodological developments of Nuclear Magnetic Resonance Spectroscopy have extended the limits in the search of biomolecular structure. The major information extracted from NMR spectroscopic data in structure determination of biomolecules is the interproton distances derived from nuclear Overhauser effect. Until recently, distance calculation from NOESY spectra were calculated via the so called two-spin approximation. Relaxation matrix analysis of NOESY data provides the best method of reducing the error introduced by the two-spin approximation. We have evaluated the accuracy of the oligonucleotide structures derived by hybrid matrix/molecular dynamics methodology. Using NOESY data simulated from the known crystal structure of the DNA dodecamer d(CGCGAATTCGCG)2 the ability of the hybrid matrix method to reproduce the crystal structure from model built A- and B-form starting geometries has been evaluated. Suitably constructed six different NOESY volume and distance constraint sets were used to examine the influence of the accuracy and the quantity of data NOESY data available. The investigation reveals that the hybrid matrix method is capable of producing oligonucleotide structures which are fat more accurate than those derived from the two-spin approximation. Since the better accuracy of inerproton distances achieved by hybrid matrix method contributed to the accuracy of the structures derived, it was observed that the availability of a larger number of NOESY volume constraints considerably improve the accuracy of structures. The hybrid matrix approach was used to determine the solution structures of a pseudo symmetric lac operator tetradecamer DNA fragment and its mutant. The hybrid matrix method successfully produced accurate structures of the two oligonucleotides. The validity of the structures were examined by comparing the experimentally determined parameters with those calculated from the derived structures. The comparison of the structures derived shows only minor differences between the two structures. This results are in accord with the observations made in the binding studies of the lac 'head Piece' to these oligonucleotides.

Degree

Ph.D.

Advisors

Gorenstein, Purdue University.

Subject Area

Biochemistry

COinS