Deoxyuridine Residues Research Articles

５位置換ピリミジンヌクレオシドを含むオリゴヌクレオチドの合成とその性質

Our recent study on the oligodeoxyribonucleotide (ODN) containing 5-substituted pyrimidine nucleosides is described. 5-Substituted 2 '-deoxyuridine derivatives and 5-substituted arabinofuranosyluracil derivatives were synthesized from 2, 2 '-anhydro-5-methoxycarbonylmethyluridine, which was synthesized from arabinoaminooxazoline and dimethyl α-bromomethylfumarate. Modified ODNs bearing these nucleoside analogs were prepared chemically by pre-synthetic modification method or post-synthetic modification method. Effect of 5-substituent groups on DNA/DNA or DNA/RNA duplexes was investigated by measuring the melting behaviors. Some of these modified ODNs are expected as antisense ODNs since these could induce RNase H activity and impart stability against nuclease. Also, 5-substituted 2 '-deoxyuridine analog triphosphates served as substrates of thermophilic family B DNA polymerases in a primer extension reaction or PCR, to give the modified ODNs. 5-Methoxycarbonylmethy1-2'-deoxyuridine residues incorporated into DNA by PCR could be used to post-synthetic derivatization. This finding is useful for in vitro selection of the functionalized DNA.

Synthesis of oligonucleotides with a 2'-cap at the 3'-terminus via reversed phosphoramidites.

[reaction: see text] A method is presented for the synthesis of single compounds or small combinatorial libraries of oligonucleotides with 2'-acylamido-2'-deoxyuridine residues at the 3'-terminus. Selection experiments identified the residue of anthraquinone-2-carboxylic acid as a "molecular cap" that increases the UV melting point of the duplex (5'-ACGCGU-3')(2) by up to 28 degrees C compared to the unmodified control duplex.

Pathways of accumulation and repair of deoxyuridine residues in DNA of higher and lower organisms.

Uracil DNA glycosylase hydrolyzes the N-glycosidic bond between sugar phosphate backbone and uracil residue appearing as the result of spontaneous deamination of cytosine or during wrong incorporation of dU residues during DNA synthesis. Uracil DNA glycosylases are very conservative enzymes. They have been recognized in all pro- and eukaryotic organisms and also in pox and herpes viruses. This review highlights the pathways of accumulation of uracil and its derivatives in DNA, the main physicochemical and biochemical properties of uracil DNA glycosylase, and regulation of its functioning. Special attention is paid to detailed mechanisms of recognition and removing of damaged (or wrong) base by uracil DNA glycosylase. These mechanisms have been validated by the methods of X-ray analysis and kinetic and thermodynamic approaches.

Iodouracil-mediated photocrosslinking of DNA to EcoRII restriction endonuclease in catalytic conditions.

We used a XeCl excimer laser with 50 ns pulses, a frequency of 0.3 Hz and a wavelength of 308 nm in appropriate conditions for the photocrosslinking of EcoRII restriction endonuclease to a 14-mer DNA duplex, containing a 5-iodo-2'-deoxyuridine residue (IdU). IdU replaced the thymidine residue within the EcoRII recognition sequence 5'-CCT/AGG. The binding of EcoRII endonuclease to the IdU-containing DNA duplex was analyzed by gel retardation assay in the presence of Ca2+ or Mg2+ ions. Photocrosslinking of EcoRII to the IdU-containing DNA duplex occurred in a pre-reactive complex formed in the presence of Ca2+ ions. Photocrosslinking yields as a function of time and UV-laser light intensity were studied.

Incorporation of a cationic aminopropyl chain in DNA hairpins: thermodynamics and hydration.

We report on the physicochemical effects resulting from incorporating a 5-(3-aminopropyl) side chain onto a 2'-deoxyuridine (dU) residue in a short DNA hairpin. A combination of spectroscopy, calorimetry, density and ultrasound techniques were used to investigate both the helix-coil transition of a set of hairpins with the following sequence: d(GCGACTTTTTGNCGC) [N = dU, deoxythymidine (dT) or 5-(3-aminopropyl)-2'-deoxyuridine (dU*)], and the interaction of each hairpin with Mg(2+). All three molecules undergo two-state transitions with melting temperatures (T(M)) independent of strand concentration that indicates their intramolecular hairpin formation. The unfolding of each hairpin takes place with similar T(M) values of 64-66 degrees C and similar thermodynamic profiles. The unfavorable unfolding free energies of 6.4-6.9 kcal/mol result from the typical compensation of unfavorable enthalpies, 36-39 kcal/mol, and favorable entropies of approximately 110 cal/mol. Furthermore, the stability of each hairpin increases as the salt concentration increases, the T(M)-dependence on salt yielded slopes of 2.3-2.9 degrees C, which correspond to counterion releases of 0.53 (dU and dT) and 0.44 (dU*) moles of Na(+) per mole of hairpin. Absolute volumetric and compressibility measurements reveal that all three hairpins have similar hydration levels. The electrostatic interaction of Mg(2+) with each hairpin yielded binding affinities in the order: dU > dT > dU*, and a similar release of 2-4 electrostricted water molecules. The main result is that the incorporation of the cationic 3-aminopropyl side chain in the major groove of the hairpin stem neutralizes some local negative charges yielding a hairpin molecule with lower charge density.

A 2‘-Acylamido Cap That Increases the Stability of Oligonucleotide Duplexes

[structure: see text]. Reported here is the synthesis of oligonucleotides containing a 2'-acylamido-2'-deoxyuridine residue at their 3'-terminus. Compared to control sequences bearing a thymidine residue, the quinolone-capped duplexes give higher UV melting points. In the case of (5'-ACGCGU-NA-2')2, where NA denotes a nalidixic acid residue, the melting point increase is up to 22 degrees C over that of (ACGCGT)2.

Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA.

High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.

The use of aptamers in large arrays for molecular diagnostics

Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). They bind tightly and specifically to target molecules; most aptamers to proteins bind with Kds (equilibrium dissociation constant) in the range of 1 pM to 1 nM. The SELEX protocol has been automated; therefore, hundreds to thousands of aptamers can be made in an economically feasible fashion. Blood and urine can be analyzed on chips that capture and quantitate proteins. SELEX has been adapted to the use of 5-bromo (5-Br) and 5-iodo (5-I) deoxyuridine residues. These halogenated bases can be specifically cross-linked to proteins. Selection pressure during in vitro evolution can be applied for both binding specificity and specific photo-cross-linkability. These are sufficiently independent parameters to allow one reagent, a photo-cross-linkable aptamer, to substitute for two reagents, the capture antibody and the detection antibody, in a typical sandwich array. After a cycle of binding, washing, cross-linking, and detergent washing, proteins will be specifically and covalently linked to their cognate aptamers. Because no other proteins are present on the chips, protein-specific stain will now show a meaningful array of pixels on the chip. Learning algorithms and retrospective studies should lead to a robust, simple, diagnostic chip.

Regeneration of 4-thio-2′-deoxyuridine residues in DNA

We report a facile, quantitative regeneration of the thiocarbonyl group from S-alkylated 4-thio-2′-deoxyuridine residues by treatment with NaSH within oligodeoxynucleotides.

Solution structure of an intramolecular DNA triplex containing 5-(1-propynyl)-2'-deoxyuridine residues in the third strand.

Incorporation of the modified base 5-(1-propynl)-2'-deoxyuridine (propynylU) in the third strand of a triplex leads to enhanced triplex stabilization. To investigate effects of the propyne nucleotide on triplex structure and the factors underlying the increased stability, we have determined the solution structure of the intramolecular DNA pyrmidine-purine-pyrimdine d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-PCPCPCPP) (PDD-EG), which contains 5-(1-propynl)-2'-deoxyuridine (P) in the third strand and hexakis(ethylene glycol) linkers [(EG)6]. The structure was calculated using X-PLOR with distance and dihedral angle restraints obtained from two-dimensional NMR experiments and refined with the direct relaxation matrix method. The structures show that the extended aromatic electron cloud of the propynylU nucleotide stacks well over the 5'-neighboring nucleotides, resulting in increased stabilization. The propynylU nucleotides also affect the overall structure of the triple helix. A comparison of the structure to that of the nonmodified intramolecular DNA triplex of the same sequence, d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-TCTCTCTT) (DDD-EG), shows that PDD-EG has a more A-DNA like X displacement and inclination than DDD-EG yet still maintains predominantly S-type sugar puckers as found in DDD-EG and other DNA triplexes.

Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I.

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.

Facile Post-Synthetic Derivatization of Oligodeoxynucleotide Containing 5-Methoxycarbonylmethyl-2′-Deoxyuridine

5-Methoxycarbonylmetyl-2′-deoxyuridine residue was incorporated into oligoDNAs containing either an exclusive thymidine residue (dT) or all four natural deoxynucleoside residues (dA, dG, dC, dT) via a phosphoramidite method. The treatment of the fully protected oligomer bound to controlled pore glass (CPG), with a variety of polyamine resulted in the release of the oligomer from CPG and the incorporation of the polyamine at the 5-position of the uracil component, simultaneously and in good yields.

Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.

Carboranyl oligonucleotides. 3. Biochemical properties of oligonucleotides containing 5-(o-carboranyl-1-yl)-2'-deoxyuridine.

Boronated oligonucleotides are potential candidates for boron neutron capture therapy, antisense technology, and as tools in molecular biology. The biological properties of dodecathymidylic acids containing one or more 5-(o-carboran-1-yl)-2'-deoxyuridine residues at different locations within the oligonucleotide chain were studied. 5-(o-Carboran-1-yl)-2'-deoxyuridine containing oligonucleotides manifested marked increased lipophilicity and resistance to 3'- or 5'-phosphodiesterases compared to the corresponding unmodified oligomer. They were substrates for T4 polynucleotide kinase and primers for Escherichia coli polymerase I and human immunodeficiency virus type 1 reverse transcriptase but not for human DNA polymerase alpha and beta. They also formed heteroduplexes that were substrates for E. coli RNase H, an essential property for antisense technology. These studies indicate that the carboranyl-containing oligonucleotides have desirable properties that need to be exploited further in the design of novel biopharmaceuticals.

The phosphodiester bond 3' to a deoxyuridine residue is crucial for substrate binding for uracil DNA N-glycosylase.

Using the method of water-soluble carbodiimide-induced chemical ligation, four 27-member oligodeoxyribonucleotides containing a pyrophosphate internucleotide bond near or adjacent to a deoxyuridine residue were prepared. Escherichia coli uracil DNA N-glycosylase (UDG) activity was found to be sensitive to the presence of an internucleotide pyrophosphate bond in both single- and double-stranded DNA. The rate of uracil excision from single-stranded DNA containing a pyrophosphate bond adjacent to the uracil residue, either 3' or 5', was 0.01% and 0.1% of the rate of uracil removal from control DNA without a pyrophosphate bond, respectively. The rate of uracil excision from duplex DNA containing a pyrophosphate bond 3' or 5' to the uracil residue was also reduced, being 0.1% and 1% the rate of uracil removal from the corresponding duplex DNA control. Placing the pyrophosphate bond one nucleotide 5' or 3' away from the deoxyuridine in both single- and double-stranded oligodeoxyribonucleotides provided much better substrates for UDG. Kinetic measurements showed that the pyrophosphate bond placed adjacent to the deoxyuridine residue drastically reduced the affinity of UDG toward the modified DNA substrate, with the greatest effect occurring when the pyrophosphate bond was 3' adjacent to the deoxyuridine. The enzyme was able to excise a 3'-terminal uracil at the nicked site of a nicked duplex, DNA, provided that the terminal deoxyuridine was 3'-phosphorylated. The effect of the pyrophosphate bond on the substrate susceptibility of oligonucleotides containing deoxyuridine is discussed with respect to the mechanism of action of UDG.

Mutagenesis in Escherichia coli by three O6-substituted guanines in double-stranded or gapped plasmids.

Plasmids were constructed with guanine (G) or O6-methyl- (m6G), O6-ethyl-(e6G), or O6-benzyl- (b6G) guanine in the initiation codon (ATG) of the lacZ' gene. Four deoxyuridine residues were incorporated near the modified guanine in the complementary strand. The deoxyuridine-containing plasmids exhibited similarly high transformation efficiencies in ung- Escherichia coli, although the frequency of mutations induced by m6G, e6G, and b6G residues was relatively low. Treatment of the plasmids with uracil-DNA glycosylase (UDG), to remove the uracil residues, or UDG and exonuclease III, to create a gap in the deoxyuridine-containing strand, reduced transformation efficiency for adduct-containing plasmids but did not affect transformation efficiency for control plasmids. However, the same treatments dramatically enhanced mutagenesis by m6G, e6G, and b6G. These results were consistent with blockage of replication by the modified guanines in double-stranded plasmids resulting in preferential replication of the complementary strand. Replication past the modified guanines was forced in the gapped plasmids. The frequency of modified guanine-induced mutations in gapped vectors was similar in strains of E. coli that were proficient in DNA polymerase III but deficient in either DNA polymerase I or II or both polymerase I and II suggesting either that polymerase III was primarily responsible for adduct bypass in all strains or that the probability of base misinsertion during bypass by either polymerase I or II was similar to that for polymerase III. Repair studies with gapped plasmids indicated that m6G was subject to repair by Ada methyltransferase and to postreplication processing by methylation-directed mismatch repair. Neither e6G nor b6G were similarly repaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Monitoring DNA dynamics using spin-labels with different independent mobilities.

The electron paramagnetic resonance (EPR) spectra of spin-labeled DNA duplexes, both bound to DEAE-Sephadex and free in solution, have been analyzed. The nitroxide spin-labels are covalently linked to a deoxyuridine residue using either a monoacetylene or diacetylene tether. This difference in tether length produces a dramatic difference in the independent mobility of the nitroxide relative to the DNA. In the case of the monoacetylene tether, the motion of the nitroxide has previously been shown to be tightly coupled to that of the DNA duplex. With the diacetylene tether, there is considerable independent motion of the probe. The diacetylene tether is intended to minimize the possibility of the nitroxide producing a perturbation of the dynamics of DNA. It is demonstrated here that, when coupled via the diacetylene tether, the nitroxide undergoes a rapid uniaxial rotation about the tether. A detailed analysis of the EPR spectrum of duplex DNA in solution, spin-labeled using the diacetylene tether, demonstrates that the motion of the nitroxide can be modeled in terms of this independent uniaxial rotation together with motion of the DNA which is consistent with the global tumbling of the duplex. As was previously found using the monoacetylene tether, there is no evidence of rapid, large-amplitude motions of the base pair in the EPR spectrum of a nitroxide coupled to duplex DNA via the diacetylene tether. This result confirms the small amplitudes of internal motion, local and collective, previously observed in duplex DNA with the monoacetylene-tethered nitroxide.

Synthesis of 3,N4-etheno, 3,N4-ethano, and 3-(2-hydroxyethyl) derivatives of 2'-deoxycytidine and their incorporation into oligomeric DNA.

3,N4-Etheno, 3,N4-ethano, and 3-(2-hydroxyethyl)derivatives of 2'-deoxycytidine arise in mammalian DNA that has been exposed to the metabolic products of either vinyl chloride or the antitumor drug bis(chloroethyl)nitrosourea. These chemically-related adducts are thought to be associated with both mutagenesis and carcinogenesis. In this paper we report reliable syntheses of these deoxynucleosides and incorporation of the latter into oligodeoxynucleotides by the phosphoramidite route, using automated methods. It was found that 3-(2-hydroxyethyl)-2'-deoxycytidine is unstable in aqueous solution and undergoes an autoinduced hydrolysis to 3-(2-hydroxyethyl)-2'-deoxyuridine. The rate of this hydrolysis was found to be pH-dependent, having a maximum around pH 8, and a half-life of approximately 5 h. At higher or lower acidities, the reaction rate falls, indicating that the process involves a general acid-base catalysis. Thus in this case, oligomers were obtained that possessed 3-(2-hydroxyethyl)-2'-deoxyuridine residues, rather than the cytidine analogue. It is likely that the former represents the longer-lived species in DNA under physiological conditions. Representative oligomers containing these chemical lesions were analyzed by mass spectrometric and enzymatic degradation methods to confirm their structures.

Analysis of post-synthetically modified oligodeoxynucleotides by electrospray ionization mass spectrometry.

Electrospray-ionization mass spectrometry is used for the effective analysis of post-synthetically modified oligodeoxynucleotides, wherein thiol groups present on 4-thio-2′-deoxyuridine residues within the oligomers are used as site-specific points of attachment for functionalized tethers.

Sequence-specific modification of DNA fragments by oligonucleotide derivatives containing alkylating groups at the C5 position of deoxyuridine.

DNA fragments are modified with high efficiency (80–90%) by alkylating derivatives of oligonucleotides bearing 4(N-2-chloroethyl-N-methylamino)benzyl group at the C5 atom of the terminal or internal deoxyuridine residue. Target modification at a single point is achieved.