5-[3-(E)-(4-Azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2′-deoxy- uridine-5′-triphosphate Substitutes for Thymidine-5′-triphosphate in the Polymerase Chain Reaction Tatyana S. Godovikova,* Dmitri M. Kolpashchikov, † Tatyana N. Orlova, Vladimir A. Richter, Tamara M. Ivanova, Sergey L. Grochovsky, ‡ Tatyana V. Nasedkina, ‡ Lubov S. Victorova, ‡ and Andrey I. Poletaev ‡ Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, 630090 Novosibirsk, Lavrentiev av. 8, Russia, Novosibirsk State University, Pirogova av. 2, 630090 Novosibirsk, Russia, and Engelhard Institute of Molecular Biology, Russian Academy of Sciences, Vavilova av. 32, 117984 Moscow, Russia. Received December 14, 1998 The DNA targets may be labeled and simultaneously amplified in the polymerase chain reaction (PCR) using a pair of respective primers after elongation with nucleoside-5′-triphosphates carrying photoreactive groups. The amplified DNA may be subsequently photoactivated by irradiation above 300 nm, resulting in photo-cross-linking of the strands. For this goal 5-[3-(E)-(4-azido-2,3,5,6- tetrafluorobenzamido)propenyl-1]-, 5-{N-[N′-(4-azido-2,3,5,6-tetrafluorobenzoyl)-3-aminopropionyl]ami- nomethyl}-, and 5-{N-[N′-(2-nitro-5-azidobenzoyl)-3-aminopropionyl]aminomethyl}-2′-deoxyuridine- 5′-triphosphate (VII, VIa, and VIb) derivatives have been synthesized. It was found that VII is capable of efficiently elongating DNA primers with both Klenow fragment DNA polymerase I and Thermus aquaticus DNA polymerase. Thereto, it turned out to provide quantitative incorporation in DNA as revealed by the formation of the full-length amplificate by PCR in the presence of this photoreactive analogue without any dilution with natural dTTP. On the contrary, it was found, that incorporation of VIa and VIb do not permit further DNA replication. INTRODUCTION Nucleoside-5′-triphosphate derivatives carrying a pho- toreactive aryl azido residue attached to heterocyclic moiety were proposed a few years ago as substrates of the nucleic acid polymerases for the preparation of photoreactive oligonucleotides. UTP and CTP analogues were used for preparation photoreactive transcript, which can be used for affinity labeling of Escherichia coli RNA polymerase (1, 2). The dCTP derivative carrying a p- perfluoroazido benzoyl residue bound to exocyclic NH 2 group via a -CH 2 CH 2 NH- spacer was prepared and used for primer extension by a DNA polymerase R-primase complex from human placenta. The duplex obtained was demonstrated to carry out affinity modification of a number of the subunits of the complex (3). The same derivative as well as another one, carrying p-perfluoro- azido benzoyl residue attached to 5-C of dCTP via the spacer -CHdCH-CH 2 -NH 2, was found to be a substrate of the HIV-1 reverse transcriptase, and it was shown that specific modification of DNA template in the complex with the enzyme was carried out (4). The primer exten- sion products obtained with 37-meric DNA and M13 DNA templates were found to photo-cross-link with both the enzyme and template DNA (4, 5). Thus, the approach turned out to be promising for the specific labeling of both polymerases and DNA templates. To expand the pos- sibilities of the approach a number of dTTP, dCTP and dATP analogues were prepared as the tools for investiga- tion of nucleic acidsnucleic acid and proteinsnucleic acid interactions (6). Recently, we proposed to use this approach to elaborate a new version of the fluorescent DNA probes localization on chromosomes and chromatin (7). This version is based upon the elongation of the hybridized probe in the presence of DNA polymerase and a mixture of deoxy- nucleoside-5′-triphosphate containing a photoreactive analogue of one of the substrates. This procedure permits the subsequent photo-cross-linking of the elongated probe with chromatin, thus making possible the following extensive washing to remove nonspecific signals and, consequently, to achieve higher contrast of the specific one. In our first work, we used a dTTP analogue with 2-nitro-5-azidobenzoyl residue attached to 5-C atom of deoxyuridine-5′-triphosphate via a -CH 2 NHC(O)(CH 2 ) 2 - NH- spacer. Obviously, the efficient incorporation of photoreactive dNTP 1 in the product of DNA replication is one of essential prerequisites for the subsequent efficient cross- linking. The main goal of this work was to prepare the photoreactive nucleoside-5′-triphosphate derivative, which can be efficiently incorporated into DNA primers by DNA polymerases with subsequent cross-linking with the template DNA under irradiation. Since it was shown earlier that, among aryl azides investigated, the p-azido perfluorobenzoyl residue is the most reactive toward complementary oligonucleotide chain (8, 9), we have * To whom correspondence should be addressed. Phone: 007- 3832-396274. Fax: (7) (3832) 333677. E-mail: silnik@ niboch.nsc.ru. † Novosibirsk State University. ‡ Engelhard Institute of Molecular Biology. 1 Abbreviations: (d)NTP, 2′-deoxyribonucleoside-5′-triphos- phates; NTP, nucleoside-5′-triphosphates; DMF, dimethylfor- mamide; TEA, triethylamine; TEAB, triethylammonium bicar- bonate; DCC, N,N′-dicyclohexylcarbodiimide; Taq DNA poly- merase, Thermus aquaticus DNA polymerase. 529 Bioconjugate Chem. 1999, 10, 529-537 10.1021/bc980144r CCC: $18.00 © 1999 American Chemical Society Published on Web 04/30/1999