NMR studies of single-site phosphate ethylated oligodeoxynucleotides.
Item
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Title
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NMR studies of single-site phosphate ethylated oligodeoxynucleotides.
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Identifier
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AAI9304642
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identifier
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9304642
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Creator
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Cahill, Sean Michael.
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Contributor
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Adviser: Michelle S. Broido
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Date
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1992
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Chemistry, Physical
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Abstract
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A series of {dollar}\sp1{dollar}H and {dollar}\sp{lcub}31{rcub}{dollar}P nuclear magnetic resonance (NMR) studies have been performed on an eight-base self-complementary oligonucleotide in its duplex state, {dollar}\{lcub}{dollar}d(GGAATTCC){dollar}\{rcub}\sb2,{dollar} and two of its single-site phosphate ethylated analogues, {dollar}(S\sb{lcub}\rm p{rcub},S\sb{lcub}\rm p{rcub}){dollar}- and {dollar}(R\sb{lcub}\rm p{rcub},R\sb{lcub}\rm p{rcub}){dollar}-{dollar}\{lcub}{dollar}d(GGA(ethyl)ATTCC){dollar}\{rcub}\sb2{dollar} in order to determine the structural and stability changes in DNA induced by phosphate alkylation and to correlate the observed changes with the stereochemistry of alkylation. The {dollar}\sp1{dollar}H chemical shift differences between the native octamer duplex and its ethylated analogues are localized to nucleotides that flank the phosphotriester lesion and are more pronounced for the {dollar}R\sb{lcub}\rm p{rcub}{dollar}-analogue which contains a phosphotriester ethyl group oriented into the major groove of the helix. Upfield shifts of {dollar}\sp{lcub}31{rcub}{dollar}P resonances corresponding to the GpA and ApT phosphodiesters and the ApA phosphotriester within the {dollar}R\sb{lcub}\rm p{rcub}{dollar}- and {dollar}S\sb{lcub}\rm p{rcub}{dollar}-analogues were observed. The {dollar}R\sb{lcub}\rm p{rcub}{dollar}-duplex has a lowered duplex stability relative to the native octamer duplex and {dollar}S\sb{lcub}\rm p{rcub}{dollar}-duplex as monitored by the temperature dependence of the imino proton linewidths. However, only small differences were observed in the base-pair lifetimes and the {dollar}\sp{lcub}31{rcub}{dollar}P spin-lattice relaxation rates between the native octamer duplex and its ethylated analogues.;In addition, a series of computer simulations were performed to evaluate the effectiveness of different structure refinement methods for determining nucleic acid conformations in solution using interproton distance data derived from NMR experiments. The computer simulations demonstrate that structure refinement methods, when used with a set of interproton distance data which mimics that derived from typical NMR experiments on nucleic acids, can accurately reproduce many of the conformational features of a target structure. Although the lack of a complete set of interproton distances prevented the use of refinement methods to generate accurate structures for the octamer duplexes, the available data suggests that the {dollar}R\sb{lcub}\rm p{rcub}{dollar}-duplex adopts a different conformation in the vicinity of the phosphotriester lesion relative to the native octamer duplex and the {dollar}S\sb{lcub}\rm p{rcub}{dollar}-duplex. Overall, the lowered duplex stability, the chemical shift differences, and the interproton distance differences of the ({dollar}R\sb{lcub}\rm p{rcub},R\sb{lcub}\rm p{rcub}{dollar})-{dollar}\{lcub}{dollar}d(GGA(ethyl)ATTCC){dollar}\{rcub}\sb2{dollar} duplex relative to the native octamer duplex and the {dollar}(S\sb{lcub}\rm p{rcub},S\sb{lcub}\rm p{rcub}){dollar}-{dollar}\{lcub}{dollar}d(GGA(ethyl)ATTCC){dollar}\{rcub}\sb2{dollar} duplex suggest that a phosphotriester moiety oriented into the major groove induces a conformational change which destabilizes DNA duplexes in solution.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
