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 (original) (raw)

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

Proceedings of the National Academy of Sciences, 1986

We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyuridine 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 coli DNA polymerase I. In in vitro DNA synthesis reactions using bacteriophage M13 single-stranded DNA as the template and 5-N3dUTP in place of dTTP, a photoactive complementary strand was synthesized by DNA polymerase I. The complementary strand was not synthesized when the 5-N3dUTP was substituted for dCTP or when it was exposed to ultraviolet light prior to the addition of DNA polymerase I. Using a synthetic lac operator template of 26 bases and a 15-base primer, we generated a photoactive 26-base-pair lac operator by enzymatically incorporating 5-N3dUMP with DNA polymerase I. Crosslinking of this photoactive DNA fragment to lac repressor was totally dependent on the presence of UV light and was reduced 78% by 150 ...

A Binary System of Photoreagents for High-Efficiency Labeling of DNA Polymerases

Biochemical and Biophysical Research Communications, 2001

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.

Reagents for Modification of Protein–Nucleic Acid Complexes: II.1Site-Specific Photomodification of Mammalian DNA Polymerase β Complexes with Primers Extended by dCTP exo-N-Substituted Arylazido Derivatives

2001

Substrate properties of the earlier synthesized and characterized dCTP derivatives bearing in the exo-N-position of cytosine 2-(4-azido-2,3,5,6-tetrafluorobenzoylamino)ethyl (I), 2-(2-nitro-5-azidobenzoylamino)ethyl (II), 2-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxymethylcarbonylamino)ethyl (III), 4-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy)butyloxy (IV), or 4-(4-azido-2,3,5,6-tetrafluorobenzylidenehydrazinocarbonyl)butylcarbonylamino (V) groups were studied in the primer extension reaction catalyzed by rat DNA polymerase ß. Unlike the earlier results obtained with HIV reverse transcriptase, dCTP derivatives (I)–(III) were not recognized by rat DNA polymerase

Site-Specific Photomodification of Single-Stranded DNA Targets by Arylazide and Perfluoroarylazide Derivatives of Oligonucleotides

Antisense and Nucleic Acid Drug Development, 1996

Highly efficient site-specific photomodification of single-stranded DNA targets was achieved with oligonu¬ cleotide reagents bearing aromatic azido groups (R (R1 = /»-azidotetrafluorobenzoyl, R2 = 2-nitro-5-azidobenzoyl, R3 = p-azidobenzoyl) at either the terminal phosphate or at the C5 position of deoxyuridine at the end or inside of the oligonucleotide chain. The extent of modification strongly depends on the reagent type. It does not exceed 5% in the case of the reagent with R3. It was 25%-50% and 60%-70% for the reagents with R2 and R1 depending on the target structure. The reagent with perfluoroarylazide group R1 appeared to be most efficient. The extent of covalent adduct formation amounts to 70% for all reagents bearing a perfluoroarylazide group at the end of the oligonucleotide chain, independently of whether it was attached to the 3'or 5'-phosphate or to the C5 of deoxyuridine. The reagents with the reactive group within the chain provided fewer cross-links (50%-55%). The reagents with R1 and R2 were found to be sensitive to the nucleotide structure of the target. Guanine and cytosine residues were modified preferentially when adjacent to the R1 or R2 group of the reagent, respectively.

Control of DNA Hybridization with Photocleavable Adducts¶

Photochemistry and Photobiology, 2005

Previous reports have shown that 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE-caged oligonucleotides have changed spectrophotometric and electrophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence-based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14-16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity.

5‑Substituted Pyrimidine and 7‑Substituted 7‑Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs

A complete series of 5-substituted uracil or cytosine, as well as 7-substituted 7-deazaadenine and 7-deazaguanine 2′-deoxyribonucleoside triphosphates (dNTPs) bearing substituents of increasing bulkiness (H, Me, vinyl, ethynyl, and phenyl) were systematically studied in competitive primer extension in the presence of their natural counterparts (nonmodified dNTPs), and their kinetic data were determined. The results show that modified dNTPs bearing π-electron-containing substituents (vinyl, ethynyl, Ph) are typically excellent substrates for DNA polymerases comparable to or better than natural dNTPs. The kinetic studies revealed that these modified dNTPs have higher affinity to the active site of the enzyme−primer−template complex, and the calculations (semiempirical quantum mechanical scoring function) suggest that it is due to the cation−π interaction of the modified dNTP with Arg629 in the active site of Bst DNA polymerase. B ase-functionalized DNA or oligonucleotides have attracted growing interest and find diverse applications. 1 Apart from chemical synthesis on solid support, they can be efficiently prepared by enzymatic synthesis using DNA polymerases and base-modified 2′-deoxyribonucleoside triphosphates (dNTPs) as substrates. 2−4 Number of methods based on the polymerase incorporations of modified nucleotides have been developed, 2 and the applications include fluorescent, 5 spin, 6 or redox 7 labeling; introduction of functional groups for DNAzymes or aptamers; 8−13 reactive groups for cross-linking 14 or even polymers, 15 oligonucleotides, 16 and protein 17 molecules; or protection of DNA against cleavage by restriction endonu-cleases 18−22 or regulation of transcription. 23 Mechanistic and structural studies showed 3,24−29 that there is a significant space in the major groove direction on the complex of polymerase, primer and template which enables efficient incorporation of dNTPs bearing even bulky substituents if they are linked to position 5 of pyrimidines or position 7 of 7-deazapurines. In most of these enzymatic methods, the polymerase reactions using a modified dNTP are performed in the absence of its natural counterpart (unmodified dNTP). Although some PCR reactions 30−32 or primer extension (PEX) 24−29 studies were reported with mixtures of natural and modified dNTPs and metabolic labeling for quantification of DNA synthesis is based on competitive incorporations of bromouracil, 33 or more recently of ethynyl-34−36 or azido-substituted 37 nucleotides (generated in vivo by intracellular phosphorylation of the corresponding nucleosides), no systematic quantitative studies of competitive incorporations of modified nucleotides in the presence of natural dNTPs has been reported, and the modified dNTPs were generally believed to be worse substrates for DNA polymerases than their natural counterparts. Recently, we reported a preliminary communication 38 on competitive incorporation of several 7-substituted-7-deazaadenine and 5-substituted cytosine dNTPs and found that 7-aryl-7-deaza-dATP analogues (dA R TPs) are significantly better substrates for DNA polymerases than dATP, whereas the corresponding 5-modified dC R TP analogues were comparable or slightly worse substrates than dCTP. Here, we report the results of the extended comprehensive and systematic study of competitive incorporations of all four modified nucleotides by various polymerases, kinetic studies, and molecular modeling in order to explain the previous surprising results (Scheme 1). ■ RESULTS AND DISCUSSION In our previous works, 18−22 we studied the influence of major-groove modifications on cleavage of DNA by Type II restriction endonucleases (REs) and found that some of these enzymes can tolerate small substituents, whereas the presence of bulkier

Isolation and characterization of pyrimidine-psoralen-pyrimidine photodiadducts from DNA

Journal of the American Chemical Society, 1982

torsional rigidity than linear DNA. It is not clear whether a direct relevance to the NMR experiment exists because the fluorescence depolarization results are quite insensitive to bending motions and also because the intercalated ethidium bromide might significantly alter the dynamic properties of the supercoiled DNA compared to its natural form. In another study, conducted by the perturbed y y angular correlation (PAC) method (Martin, P. W.; El-Kuteb, S.; Kuhnlein, U. J . Chem. Phys. 1982, 76, 3819-3822) on supercoiled PM2 DNA, and apparent isotropic correlation time of 8.2 f 0.6 ns was measured, compared to 62 (+12/-10 ns) for presumably much more flexible single-stranded DNA (Kolfas, C. A.; Sideris, E. G.; El-Kuteb, S.; Martin, P. W.; Kuhnlein, U.

Sequence-specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-(α]-thymidylate covalently linked to an azidoproflavine derivative

Nucleic Acids Research, 1987

A 3-azidoproflavine derivative was covalently linked to the 5'-end of an octathymidylate synthesized with the [a]-anomers of the nucleoside. Two target nucleic acids were used for this substituted oligo-[a]-thymidylate : a 27-mer single-stranded DNA fragment containing an octadeoxyadenylate sequence and a 27-mer duplex containing eight contiguous A.T base pairs with all adenines on the same strand. Upon visible light irradiation the octa-[a]-thymidylate was photocrosslinked to the single-stranded 27-mer. Chain breaks were induced at the crosslinked sites upon piperidine treatment. From the location of the cleavage sites on the 27-mer sequence it was concluded that a triple helix was formed by the azidoproflavine-substituted oligo-[a]-thymidylate with its complementary oligodeoxyadenylate sequence. When the 27-mer duplex was used as a substrate cleavage sites were observed on both strands after piperidine treatment of the irradiated sample. They were located at well defined positions which indicated that the octathymidylate was bound to the (dA)8.(dT)8 sequence in a parallel orientation with respect to the (dA)8-containing strand. Specific binding of the [a]-octathymidylate involved local triple strand formation with the duplex (dA)8.(dT)8 sequence. This result shows that it is possible to synthesize sequence-specific molecules which specifically bind oligopurine-oligopyrimidine sequences in double-stranded DNA via recognition of the major groove hydrogen bonding sites of the purines.