Bioconjugate chemistry

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

PMID 9177841


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'-ETTTTMeCTTTTMeCMeCTTTMeCTTTT-3' at 37 degrees 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 N7 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 k1 of (2.7 +/- 0.5) x 10(-5) S(-1) (t1/2 = 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 k2 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.

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2-Bromoacetamide, 98%