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Nucleic Acids Research, 2000, Vol. 28, No. 21 4382-4390
© 2000 Oxford University Press

Two novel dATP analogs for DNA photoaffinity labeling

Martin Zofall and Blaine Bartholomew*

Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Room 229C, Neckers Building, 1245 Lincoln Drive, Carbondale, IL 62901-4413, USA

Two new photoreactive dATP analogs, N6-[4-azidobenzoyl–(2-aminoethyl)]-2'-deoxyadenosine-5'-triphosphate (AB-dATP) and N6-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2'-deoxyadenosine-5'-triphosphate (DB-dATP), were synthesized from 2'-deoxyadenosine-5'-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N1-(2-aminoethyl)-2'-deoxyadenosine-5'-monophosphate to N6-(2-aminoethyl)-2'-deoxyadenosine-5'-monophosphate (N6-dAMP). Next, N6-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N6-(2-aminoethyl)-2'-deoxyadenosine-5'-triphosphate (N6-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N6-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.

* To whom correspondence should be addressed. Tel: +1 618 453 6437; Fax: +1 618 453 6440; Email: bbartholomew@siumed.edu


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