Kousuke Sato

Selective Detection of 5-Formyl-2'-deoxyuridine, an Oxidative Lesion of Thymidine, in DNA by a Fluorogenic Reagent

DNA in living cells is damaged by reactive oxygen species derived from UV light, ionizing radiation, and cellular respiration. 5-Formyl-2’-deoxyuridine (fodUrd), an oxidized thymidine lesion generated in yields comparable to that of 2’deoxy-8-oxoguanosine, induces mutation in DNA (AT-toGC transition) through the mispairing of an ionized form of fodUrd with 2’-deoxyguanosine during DNA replication. It appears that the formation of fodUrd may cause carcinogenicity and/or aging of cells. A selective and more convenient method for detecting fodUrd would be highly desirable, since the existing methods are complicated, involve time-consuming analysis by HPLC and/or mass spectrometry following complete enzymatic hydrolysis of the target DNA, and require isotope-labeled fodUrd (C and/or N) as an internal standard. Some other approaches have been developed for the detection of DNA damage. Molecular recognition through the formation of a base pair between an oligonucleotide probe containing a synthetic complimentary nucleobase and O-benzyl-dG was developed by Gong and Sturla. A common method is to detect DNA damage with enhanced reactivity by selective tagging with a molecule that contains a reporter group. Ide et al. first demonstrated the detection of abasic sites by the use of an aldehyde reactive probe. Other selective detection methods with chemical reagents have recently been reported. However, the signal-to-noise (S/N) ratio is a problem in these methods, as the fluorescent and/or colorimetric molecules used have higher background signals. Herein, we report a new concept for the simple detection of fodUrd in damaged DNA with a fluorogenic reagent. The reagent 2-amino-4,5-dimethoxythiophenol (3’) shows no fluorescence before reaction with the target fodUrd in DNA. However, upon the reaction of 3’ with fodUrd, the formyl group at the 5-position of fodUrd is converted into a benzothiazol-2-yl group, which is directly conjugated with the uracil group. This ring system is similar to luciferin, which undergoes the luciferase reaction to produce luminescence. Several 5-heteroaryl 2’-deoxyuridines with good fluorescence properties have been reported. Thus, fodUrd in DNA could be detected directly by fluorescence measurement without enzymatic hydrolysis of the target DNA to its corresponding nucleosides, HPLC separation, and analysis. First, we synthesized the reagent bis(4,5-dimethoxyanilin2-yl)disulfide (3) from 3,4-dimethoxyaniline (1) in two steps (Scheme 1). Since oxidation by air leads to the spontaneous dimerization of reagent 3’, the disulfide 3 was reduced to 3’ by the addition of dithiothreitol (DTT) just before treatment with fodUrd. Oxidation with H2O2 in the presence of

Incorporation of 2'-deoxy-2'-isonucleoside 5'-triphosphates (iNTPs) into DNA by A- and B-family DNA polymerases with different recognition mechanisms.

Recently, α‐L‐threofuranosyl nucleoside 3′‐triphosphates (tNTPs) have been reported to be incorporated into DNA by DNA polymerases. Isonucleosides especially the 2′‐deoxy‐2′‐isonucleosides, would be considered regioisomers of α‐L‐threofuranosyl nucleosides. Therefore, we investigated the synthesis of 2′‐deoxy‐2′‐isonucleoside 5′‐triphosphates (iNTPs) having the four natural nucleobases and their incorporation into primer–template duplexes consisting of oligonucleotides containing natural 2′‐deoxyribonucleosides and 2′‐deoxy‐2′‐isonucleosides by using primer‐extension reactions. We found that Klenow fragment (exo‐; an A‐family DNA polymerase) has strict recognition of the shape of nucleoside 5′‐triphosphates and Therminator (a B‐family DNA polymerase) has strict recognition of the shape of primer–template complexes, especially two base pairs upstream of the primer 3′ terminus.

Effects of a High‐Affinity Antibody Fragment on DNA Polymerase Reactions Near a (6–4) Photoproduct Site

Abstract— Pyrimidine (6–4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6–4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41‐mer DNA containing a (6–4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases α, β and δ (pol α, pol β and pol δ). None of these DNA polymerases were able to bypass the (6–4) photoproduct and elongation terminated at or near the 3′‐pyrimidone of the photoproduct, depending on the dNTP concentration. When a single‐chain Fv (scFv) with high affinity for the (6–4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3′‐pyrimidone by Klenow fragment, pol α, pol β or pol δ, respectively. These results suggest that the scFv can bind to the template DNA containing a (6–4) photoproduct and inhibit extension reactions by polymerases.

Highly Fluorescent 5‐(5,6‐Dimethoxybenzothiazol‐2‐yl)‐2′‐Deoxyuridine 5′‐Triphosphate as an Efficient Substrate for DNA Polymerases

We herein describe the synthesis of fluorescent 5‐(5,6‐dimethoxybenzothiazol‐2‐yl)‐2′‐deoxyuridine 5′‐triphosphate (dbtUTP) and primer extension reactions using dbtUTP. We also carried out primer extension reactions using the btU template. B family DNA polymerases, such as KOD, Deep Vent (exo‐), and 9°Nm DNA polymerases, were effective for elongation with dbtUTP. Deep Vent (exo‐) and KOD DNA polymerases have excellent fidelity for incorporating dbtUTP only at the site opposite the adenine template and only dATP when using the btU template. Therefore, dbtUTP is an excellent fluorescent nucleotide that can be incorporated into DNA by DNA polymerases.

Synthesis of 5-formyl-2'-deoxyuridine and its incorporation into oligodeoxynucleotides.

A straightforward, efficient method for the synthesis of 5‐formyl‐2′‐deoxyuridine (dfU) and solid‐phase synthesis of oligodeoxynucleotides containing dfU using a phosphoramidite method are described. The synthesis of dfU is achieved by oxidation of the 5‐methyl group in thymidine derivatives. However, incorporation of the dfU 3′‐O‐phosphoramidite into oligodeoxynucleotides proceeds in low yield, due to instability of the 5‐formyl group under conditions used for automated DNA synthesis. Therefore, oligodeoxynucleotides containing a 5‐(1,2‐dihydroxyethyl)uracil derivative are first prepared and finally oxidized by periodate to give the desired oligodeoxynucleotides containing 5‐formyluracil. Curr. Protoc. Nucleic Acid Chem. 35:1.21.1‐1.21.19.

Interactions of High Affinity Anti (6-4) Photoproduct Antibody Fragments with Damaged DNA

Abstract The interactions between chemically synthesized DNA fragments containing a T(6-4)T and antigen binding fragments (Fab) or single-chain antibodies (scFv) were investigated by X-ray crystallography, NMR, and surface plasmon resonance. The high affinity scFv protein was found to bind to the template DNA near the (6-4) photoproduct site and to interfere with DNA polymerase reactions in vitro.

Solid-Phase Modular Synthesis of Park Nucleotide and Lipids I and II Analogues

A solid-phase synthesis of Park nucleotide as well as lipids I and II analogues, which is applicable to the synthesis of a range of analogues, is described in this work. This technique allows highly functionalized macromolecules to be modularly labeled. Multiple steps are used in a short time (4 d) with a single purification step to synthesize the molecules by solid-phase synthesis.