A Novel Combined Chemical–Enzymatic Synthesis of Cross-Linked DNA Using a Nucleoside Triphosphate Analogue

Marlon Cowart, Stephen Benkovic

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

A novel method using combined chemical and enzymatic reactions to allow the preparation of covalently cross-linked DNA duplexes has been described. The method can be used to specifically link two complementary bases of a DNA duplex containing all four natural bases. The modified nucleotide 9-(2-deoxy-5-O-triphospho-β-D-ribofuranosyl)-N6,N6-ethano-2,6-diaminopurine (6edDTP) was prepared by total chemical synthesis and was found to be incorporated into DNA duplexes in the place of 2′-deoxyguanosine 5 ′-O-triphosphate by the Klenow fragment of Escherichia coli DNA polymerase I, T4 and T7 DNA polymerases, avian myeloma virus reverse transcriptase, and rat DNA polymerase β. Once incorporated, the aziridine of the nucleotide is rapidly opened by the N4 of the cytosine on the complementary strand to give cross-linked DNA, where the modified nucleotide is covalently joined to the complementary base by an ethano linkage. The duplexes produced were found to be recognized as substrates by various DNA polymerases. The Km for the incorporation of the 6edDTP into DNA catalyzed by the Klenow fragment of E. coli DNA polymerase I was found to be 29 μM, and the kcat was found to be 0.014 s−1. The modified nucleoside also served as a substrate for terminal deoxynucleotidyltransferase, where it was added to single-stranded DNA and then hybridized to a complementary strand, after which cross-linking of the two strands occurred within 1 min.

Original languageEnglish (US)
Pages (from-to)788-796
Number of pages9
JournalBiochemistry
Volume30
Issue number3
DOIs
StatePublished - Jan 1 1991

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DNA Polymerase I
Nucleosides
DNA-Directed DNA Polymerase
DNA
Nucleotides
Escherichia coli
DNA Nucleotidylexotransferase
Single-Stranded DNA
Cytosine
RNA-Directed DNA Polymerase
Substrates
Viruses
Rats
triphosphoric acid
9-(2-deoxy-5-O-triphospho-beta-ribofuranosyl)-N(6),N(6)-ethano-2,6-diaminopurine

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Cowart, Marlon ; Benkovic, Stephen. / A Novel Combined Chemical–Enzymatic Synthesis of Cross-Linked DNA Using a Nucleoside Triphosphate Analogue. In: Biochemistry. 1991 ; Vol. 30, No. 3. pp. 788-796.
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A Novel Combined Chemical–Enzymatic Synthesis of Cross-Linked DNA Using a Nucleoside Triphosphate Analogue. / Cowart, Marlon; Benkovic, Stephen.

In: Biochemistry, Vol. 30, No. 3, 01.01.1991, p. 788-796.

Research output: Contribution to journalArticle

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