Structure refinement of covalently modified oligonucleotides by two-dimensional NMR and restrained molecular dynamics
Abstract
Sequence specific modification of functionally significant target sites of genes provides an exciting avenue for the control of cellular function. The utilization of highly reactive chemical moieties attached to complimentary DNA sequences has been of special interest in this regard. Of these the 4- (N-methyl-N-(2-chloroethyl)amino) benzyl-5$\sp\prime$-phosphamido derivatives have been studied extensively. However up to date no structural studies of covalent adducts formed by these derivatives have been undertaken. This thesis describes the computation of the macromolecular structure of a covalent adduct (CA) formed between the heptanucleotide derivative 4- (N-methyl-N-(2-chloroethyl)amino) benzyl-5$\sp\prime$-phosphamido-dCCAAACA and the target sequence dTGTTTGGC. A combination of 2D NMR, hybrid relaxation matrix methods and molecular dynamics was used for the investigation. Analysis of the final structures indicated that the modification group was located close to the "vacant" site opposite the target sequence at a position that is nearly parallel to the adjacent bases of the same strand. Extensive perturbations of the backbone dihedrals were observed with $B\sb{\Pi}$ type conformations localized near the adduct formation site. To understand the reaction of CA formation, an analog of the reaction intermediate was also studied. Results of this investigation indicated that the interatomic distance between reactive centers in the CA formation reaction is approximately 8A in the averaged final structure there by explaining the low yields obtained in CA formation reactions. A structural investigation of a 14 base pair CRE binding DNA was also undertaken as part of this thesis project. Similar to CA, 2D NMR Hybrid relaxation methods in combination with MD were used for structural investigations.
Degree
Ph.D.
Advisors
Gorenstein, Purdue University.
Subject Area
Biochemistry|Biophysics|Molecular biology
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