5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA.

Abstract

We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyurldine 5'-triphosphate (5-N3dUTP) and used it to synthesize light-sensitive DNA by enzymatic incorporation. In the absence of ultraviolet light, 5-N3dUTP is a substrate for Escherichia coil DNA polymerase I. In in vitro DNA synthesis reactions using bacteriophage M13 MATERIALS AND METHODSSynthesis of 5-N3dUTP and [y-32P]5-N3dUTP. 5-Azidodeoxyuridine monophosphate (5-N3dUMP) was synthesized from 5-aminodeoxyuridine monophosphate (5-NH2dUMP). The detailed synthesis of 5-NH2dUMP, as well as the identification of the 5-N3dUMP product, will be described elsewhere. Briefly, the synthesis of 5-N3dUTP was as follows: 5.0 ml of 1 M HCl containing 10 Amol of 5-NH2dUMP was placed on ice and stirred for 15 min. Then, a solution of 10 pumol of NaNO2 in 3.0 ml of H20 was added while stirring at 00C. After 2 min, 1.5 ml of 4 M NaN3 was added with rapid stirring. After 5 min, the reaction mixture was removed from the ice bath and allowed to warm to room temperature over a 30-min period. Then, the reaction mixture was neutralized with NH40H and desalted on a Sephadex G-10 column (40 cm x 1.5 cm) equilibrated with distilled water. The first UVabsorbing fraction from the column was applied to a benzyl-DEAE (BD) cellulose column (30 cm x 1.5 cm) and eluted with a 400-ml linear gradient of 10 mM to 0. 5382The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Interferon-alpha regulates nuclear translocation and DNA-binding affinity of ISGF3, a multimeric transcriptional activator.

“…A uniformly 32P-labeled ISRE oligonucleotide containing 5-azido-deoxyuridine (Evans et al 1986;Evans and Haley 1987) was incubated with appropriate fractions, and DNA-protein complexes were resolved by gel shift (Fig. 5A).…”

Chemical approaches to the elucidation of template biosynthesis: study of replication and reverse transcription

“…Ura-5 ± Modification of DNA polymerase I from E. coli in the absence of a template ± primer duplex 24 Ura-5 Substrate of reverse transcriptase Modification of p66 and p51 from HIV-1 instead of dTTP 15 Ura-5 Substrate of reverse transcriptase Modification of p66 (ddUTP) from HIV-1 instead of dTTP 20…”

Nucleic acid-incorporated azidonucleotides: Probes for studying the interaction of RNA or DNA with proteins and other nucleic acids

“…The azidoadenosines and, more recently, 5-azidouridine (5N3U) and 2,6-diazidopurine (2,6diN3R) nucleotides, have all been used for incorporation into RNA (Cartwright and Hutchinson, 1980;Wower et al, 1988Wower et al, , 1989Wower et al, ,1990Wower et al, ,1993aSylvers et al, 1989Sylvers et al, , 1992Dontsova et al, 1992b). Similarly, 5NsdUTP and 8NsdATP have been synthesized and incorporated into DNA (Evans et al, 1986; Evans and Haley, 1987;Lee et al, 1991;Meffert and Dose, 1988;Meffert et al, 1990).…”

“…To date, the incorporation of azidodeoxynucleotides into DNA has been accomplished exclusively by enzymatic polymerization with DNA polymerase I (Evans et al, 1986; Evans and Haley, 1987;Lee et al, 1991;Meffert and Dose, 1988;Meffert et al, 1990). However, since DNA fragments and deoxynucleotide 3',5'-bisphosphates can be used as unconventional substrates for T4 RNA ligase (Brennan et al, 1983), products of enzymatic or chemical DNA synthesis can be used in a "synthesize and paste" approach to generate site-specific azidonucleotide-substituted DNA molecules.…”