Two novel dATP analogs for DNA photoaffinity labeling
AUTOR(ES)
Zofall, Martin
FONTE
Oxford University Press
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=113131Documentos Relacionados
- Test of the potential of a dATP surrogate for sequencing via MALDI-MS.
- Novel biotinylated nucleotide--analogs for labeling and colorimetric detection of DNA.
- Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.
- Cytochrome c binding to Apaf-1: The effects of dATP and ionic strength
- Enzymatic synthesis of deoxyribonucleotides, 8. The effects of ATP and dATP in the CDP reductase system from E. coli.