Biophysical properties of modified oligodeoxynucleotides.
Item
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Title
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Biophysical properties of modified oligodeoxynucleotides.
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Identifier
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AAI9000725
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identifier
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9000725
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Creator
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Quartin, Robin Sandra.
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Contributor
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Adviser: James G. Wetmur
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Date
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1989
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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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Biology, General | Chemistry, Biochemistry | Biology, Molecular
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Abstract
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The thermodynamic and biochemical properties of two types of modified oligodeoxynucleotides were investigated in order to improve upon current applications and to develop new molecular biology tools.;Oligodeoxynucleotides containing uncharged, chiral methylphosphonate linkages bound specifically to immobilized complementary DNA. These oligodeoxynucleotides were used to examine the effect of reduced charge on the thermodynamics of binding to complementary DNA or complementary oligodeoxynucleotides. The free energy decrement per linkage was {dollar}-0.75{dollar} kcal/mol in high salt. The absence of a charge change for nearest neighbor base pairs containing a methylphosphonate linkage led to greater hybrid stability for substituted oligodeoxynucleotides at low salt. Finally, analysis of dissociation temperatures indicated that substitution of methylphosphonate linkages at high salt only affected the reverse rate constant.;Oligodeoxynucleotides with different arrangements of diester and methylphosphonate linkages were examined for nuclease sensitivity in vitro and in tissue culture using an assay for the ability to gel-shift a labeled complementary phosphodiester oligodeoxynucleotide. Both 5{dollar}\sp\prime{dollar} and 3{dollar}\sp\prime{dollar} exonuclease function was impaired by methylphosphonate linkages. The smallest span of internal phosphodiester linkages correlated with the greatest resistance to endonuclease. However, in cell culture the half-lives of these oligodeoxynucleotides were independent of the number of contiguous phosphodiester linkages. RNase H assays indicated that a minimal span of three internal phosphodiester linkages in a methylphosphonate-substituted oligodeoxynucleotide was needed to allow cleavage of the RNA in the duplex.;We have also developed a highly sequence-specific capture reaction based on increased DNA-DNA hybrid stability due to substitution of bromodeoxycytidine (BrdC) for deoxycytidine (dC). BrdC-containing oligodeoxynucleotides displaced dC-containing strands from duplexes with blunt-ends or 3{dollar}\sp\prime{dollar}-overhangs. A BrdC-containing oligodeoxynucleotide used for transient sequence-specific invasion at a particular PstI site, was captured using DNA ligase and a linker oligodeoxynucleotide at over 300 times the rate for an unrelated PstI site. Incorporation of an incorrect nucleotide into a displacer strand demonstrated that branch migration terminated at a mismatch. A branched, BrdC-containing ligated capture reaction product was cloned and sequenced.
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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.
