Structural Studies of Synthetic Base Analogs in Oligodeoxynucleotides

Abstract

To examine the effect of major groove electrostatics on the stability and structure of DNA, in the first portion of this dissertation a detailed structural characterization of the 7-deaza-2'-deoxyadenosine:dT base pair in the Dickerson-Drew dodecamer (DDD) has been completed. The crystal structure of the 7-deaza-dA:dT base pair is determined at 1.1 Å in the presence of Mg2+. The 7-deaza-dA, which is isoteric for dA, has minimal effect on the base pairing geometry and the conformation of the DDD. There is no major groove cation association with the 7 deaza-dA heterocycle. NMR studies conducted as a function of temperature show increased exchange between the thymine N3 imino proton of the Y6•T7 base pair and water, suggesting reduced stacking interactions and an increased rate of base pair opening for the 7-deaza-dA:dT base pair. This correlates with the thermodynamic destabilization of the 7-deaza-dA modified duplex relative to the DDD. The second portion of this dissertation focuses upon understanding the structural basis by which the 2'-deoxynucleoside containing the synthetic base dPer recognizes the O6 benzyl-2'-deoxyguanosine nucleoside (O6-Bn-dG) in DNA, formed by exposure to N benzylmethylnitrosamine. A combination of crystallographic and NMR spectroscopic experiments reveal how dPer distinguishes between dG and O6 Bn-dG in DNA. The structure of the modified DDD in which O6-Bn-dG is placed opposite dPer to form the modified DDD-XY duplex reveals that dPer intercalates into the DNA and adopts the syn conformation about the glycosyl bond. This provides a binding pocket that allows the benzyl group of O6-Bn-dG to intercalate between dPer and thymine of the opposite strand. NMR data suggest that a similar intercalative recognition mechanism applies in solution. In solution the benzyl ring of O6 Bn-dG undergoes rotation on the NMR time scale. In contrast, structural analysis of the modified DDD in which dG is placed opposite dPer to form the DDD-GY duplex reveals that dPer adopts the anti conformation about the glycosyl bond and forms a less stable wobble pairing interaction with guanine.