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Kinetic analysis of sequence-specific alkylation of DNA by pyrimidine oligodeoxyribonucleotide-directed triple-helix formation

Taylor, Matthew J. and Dervan, Peter B. (1997) Kinetic analysis of sequence-specific alkylation of DNA by pyrimidine oligodeoxyribonucleotide-directed triple-helix formation. Bioconjugate Chemistry, 8 (3). pp. 354-364. ISSN 1043-1802. https://resolver.caltech.edu/CaltechAUTHORS:20160510-102059723

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Abstract

Attachment of a nondiffusible bromoacetyl electrophile to the 5-position of a thymine at the 5'-end of a pyrimidine oligodeoxyribonucleotide affords sequence-specific alkylation of a guanine base in duplex DNA two base pairs to the 5'-side of a local triple-helical complex. Products resulting from reaction of 5'-^ETTTT^(Me)CTTTT^(Me)C^(Me)CTTT^(Me)CTTTT-3' at 37 °C with a 29 base pair target duplex are determined by a gel mobility analysis to be oligonucleotides terminating in 5'- and 3' -phosphate functional groups, consistent with a mechanism involving alkylation, glycosidic bond cleavage, and base-promoted strand cleavage. The guanine-(linker)-oligonucleotide conjugate formed upon triple-helix-mediated alkylation at the N7 position of a guanine base in a 60 base pair duplex was identified by enzymatic phosphodiester hydrolysis of the alkylation products followed by reversed phase HPLC analysis. To determine the rate enhancement achieved by oligonucleotide-directed alkylation of duplex DNA, a comparison of rates of alkylation at N^7 of guanine in double-stranded DNA by the N-bromoacetyloligonucleotide and 2-bromoacetamide was performed by a polyacrylamide gel assay. The reaction within the triple-helical complex on a restriction fragment was determined at 200 nM N-bromoacetyloligonucleotide to have a first-order rate constant k_1 of (2.7 ± 0.5) x 10^(-5) S^(-1) (t_(1/2) = 7.2 h). The reaction of 2-bromoacetamide with a 39 base pair duplex of sequence corresponding to the restriction fragment targeted by triple-helix formation was determined to have a second-order rate constant k_2 of (3.6 ± 0.3) x 10^(-5) M^(-1) S^(-1). A comparison of the first-order and second-order rate constants for the unimolecular and bimolecular alkylation reactions provides an effective molarity of 0.8 M for bromoacetyl within the triple-helical complex.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bc970035xDOIArticle
http://pubs.acs.org/doi/abs/10.1021/bc970035xPublisherArticle
ORCID:
AuthorORCID
Dervan, Peter B.0000-0001-8852-7306
Additional Information:© 1997 American Chemical Society. Publication Date (Web): May 28, 1997. Abstract published in Advance ACS Abstracts, April 15, 1997. We are grateful for generous support from the National Institutes of Health (GM-35724).
Funders:
Funding AgencyGrant Number
NIHGM-35724
Issue or Number:3
Record Number:CaltechAUTHORS:20160510-102059723
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160510-102059723
Official Citation:Kinetic Analysis of Sequence-Specific Alkylation of DNA by Pyrimidine Oligodeoxyribonucleotide-Directed Triple-Helix Formation Matthew J. Taylor and Peter B. Dervan Bioconjugate Chemistry 1997 8 (3), 354-364 DOI: 10.1021/bc970035x
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:66880
Collection:CaltechAUTHORS
Deposited By: Victoria Brennan
Deposited On:18 May 2016 19:22
Last Modified:26 Nov 2019 11:15

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