Nucleic Acids Research 2000-11-01

Two novel dATP analogs for DNA photoaffinity labeling.

M Zofall, B Bartholomew

文献索引：Nucleic Acids Res. 28 , 4382-4390, (2000)

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摘要

Two new photoreactive dATP analogs, N(6)-[4-azidobenzoyl-(2-aminoethyl)]-2'-deoxyadenosine-5'-triphospha+ ++ te (AB-dATP) and N(6)-[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 N(1)-(2-aminoethyl)-2'-deoxyadenosine-5'-monophosphate to N(6)-(2-aminoethyl)-2'-deoxyadenosine-5'-monophosphate (N(6)-dAMP). Next, N(6)-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 N(6)-(2-aminoethyl)-2'-deoxyadenosine-5'-triphosphate (N(6)-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. N(6)-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.