Template-directed DNA photoligation via alpha-5-cyanovinyldeoxyuridine

[reaction: see text] We describe an efficient template-directed photoligation of oligodeoxynucleotides (ODNs) using alpha-5-cyanovinyldeoxyuridine (alpha(C)U). An efficient photoligation was produced by photoirradiation of an ODN containing alpha(C)U at the 3' end with an ODN containing thymine at the 5' end in the presence of a template ODN. This photoligation method is a new and efficient way to synthesize branched ODNs.     

Template-directed photoreversible ligation of DNA via 7-carboxyvinyl-7-deaza-2′-deoxyadenosine

An efficient template-directed photoligation of oligodeoxynucleotide (ODN) using 7-deaza-2′-deoxyadenosine derivative ^VZA is described. When ODN containing ^VZA at the 5′ end was photoirradiated with ODNs containing a pyrimidine base at the 3′ end in the presence of template ODN, rapid and efficient ligation (cycloaddition reaction) was observed without any byproduct formation. ODNs containing ^VZA showed an extremely high reactivity as compared with those reported in previous photoligations.     

Autonomous DNA computing machine based on photochemical gate transition

We report the construction of a one-pot autonomous DNA computing machine based on photochemical gate transition (photocleavage, hybridization, and photoligation), and we performed binary digit additions using this machine. In our method, both photochemical DNA manipulations previously reported, photoligation via 5-carboxyvinyldeoxyuridene (cvU) containing ODN and photocleavage via carbazole-modified ODN, were employed. The binary digit additions were autonomously carried out by one-time irradiation at 366 nm in the single test tube. The fluorescence readout by the DNA chip was in good agreement with the correct answer of binary digit additions. We believe that this system is easily applicable to correlation analysis between SNPs as well as other binary digit processing, such as subtraction.     

A light-controlled reversible DNA photoligation via carbazole-tethered 5-carboxyvinyluracil

We describe a light-controlled template-directed reversible DNA photoligation via carbazole-tethered 5-carboxyvinyluracil. Carbazole-tethered 5-carboxyvinyl-2'-deoxyuridine-containing oligodeoxynucleotide (ODN) can be ligated by irradiation at 366 nm in the presence of template ODN, and the ligated ODN can be split by irradiation at 366 nm in the absence of template ODN.     

Unique charge transfer properties of the four-base pi-stacks in Z-DNA

To investigate the photoreactions of BrU in Z-DNA, the photoirradiation of 5'-d(C1G2C3G4BrU5G6C7G8)-3'/5'-d(C9mG10C11A12C13mG14C15G16)-3'(ODN 1-2) was investigated. In accord with previous observations, B-form ODN 1-2 with the 5'-GBrU sequence showed very weak photoreactivity. However, Z-form ODN 1-2 in 2 M NaCl underwent photoreaction to afford 5'-d(CGC)rGd(UGCG)-3' together with the formation of imidazolone (Iz) contained 5'-d(CIzCACmGCG)-3'. The results clearly indicate that structural changes caused by the B-Z transition dramatically increased the photoreactivity of ODN 1-2. Inspection of the molecular structure of Z-DNA suggests that there is unique four-base pi-stacks at the G4-BrU5-C11-mG10 in ODN 1-2. These results suggest that the intriguing possibility that the mG10 in a complementary strand located at the end of the four-base pi-stacks may act as an electron donor. To test the hypothesis of interstrand charge transfer from mG10 to BrU5 within the four-base pi-stacks in Z-DNA, ODN 1-3 samples in which the putative donor G10 residue was replaced with 8-methoxyguanine (moG) were prepared, since moG is known to trap cation radicals to yield Iz moieties in DNA. Photoirradiation of ODN 1-3 efficiently produced 5'-d(CGC)rGd(UGCG)-3' together with formation of 5'-d(CIzCACmGCG)-3'. These results clearly indicate that the interstrand charge transfer from mG10 to BrU5 initiates the photoreaction. In clear contrast, other replacements of G with moG did not enhance the photoreactivity. The present study revealed the presence of unique four-base pi-stacks in Z-DNA and photoirradition of BrU in Z-DNA causes efficient electron transfer from G within this cluster.     

Template directed reversible photochemical ligation of oligodeoxynucleotides

We demonstrated that 5-vinyldeoxyuridine ((V)U) and 5-carboxyvinyldeoxyuridine ((CV)U) can be used to photoligate a longer oligonucleotide (ODN) from smaller ODNs on a template. By performing irradiation at 366 nm, these artificial nucleotides make photoligated ODNs with high efficiency without any side reactions. Moreover, by performing irradiation at 312 nm, these photoligated ODNs were reversed to the original ODN. (V)U needs to be irradiated 366 nm for 6 h, but (CV)U needs to be irradiated at 366 nm for 15 min. Finally, we made a self-assembled structure with an ODN containing (CV)U and observed the photoligated ODN by photoirradiation.     

New base pairing motifs. The synthesis and thermal stability of oligodeoxynucleotides containing imidazopyridopyrimidine nucleosides with the ability to form four hydrogen bonds

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.     

Electropolymerisable pyrrole-oligonucleotide: synthesis and analysis of ODN hybridisation by fluorescence and QCM

Conducting polymer films, such as polypyrrole, appear particularly attractive for the immobilisation of biological molecules by entrapment or covalent grafting. We describe here a new pyrrole phosphorarnidite building block allowing the synthesis of oligonucleotide (ODN) bearing a pyrrole moiety. The electropolymerisable pyrrole moiety was then introduced on the 5' end of the oligonucleotide. The electrosynthesis of a copolymer, from solutions containing pyrrole and pyrrole-ODN, gives in one step strongly adhesive films containing ODN probes at electrode surfaces. In this contribution, we have used such a methodology to verify its feasibility for the modification of quartz crystal microbalance (QCM) electrodes. The obtained biosensors enable the detection of DNA hybridisation in real time by micro-gravimetric transduction. Finally, as DNA targets were previously modified by biotin, we have used the affinity between biotin and avidin to validate the effectiveness of QCM transduction by fluorescence microscopy and to amplify the recorded micro-gravimetric signal.     

Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene

Two 17-mer oligodeoxynucleotide-5'-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was 3'G G T A G T T A T T C C T T C T T5'. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH2)3OPO(OH)- linker, to the 5'-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate the linker was -(CH2)5CONH(CH2)6OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, 5'T G A C C A T C A A T A A G14 G A A G18 A A G21 C C C T T C A G C G G C C3' 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.     

The effect of local structural disruption on the yield of photochemical ligation between anthracene-oligonucleotide conjugates.

The techniques of chemical ligation have attracted great attention as an alternative to enzymatic joining of DNA ends. Here we introduce the photoligation of anthracene-modified ODN conjugates through anthracene cyclodimer formation. The effect of the positions and the kinds of single base mismatch on the template was evaluated using eight templates with one-base displacements. We found out that the yield of the ligation was affected by mispairing in a position-dependent manner. Such results would be attributed to the disruption of the local structure at the ligation site.     

Synthesis and properties of trans-3',4'-bridged nucleic acids having typical S-type sugar conformation

The synthesis of nucleoside analogues with a conformationally restricted sugar moiety is of great interest. The present research describes the synthesis of BNA (bridged nucleic acid) monomers 1 and 2 bearing a 4,7-dioxabicyclo[4.3.0]nonane skeleton and a methoxy group at the C2' position. Conformational analysis showed that the sugar moiety of these monomers is restricted in a typical S-type conformation. It was difficult to synthesize the phosphoramidite derivative of the ribo-type monomer 1, while the phosphoramidite of the arabino-type monomer 2 was successfully prepared and incorporated into oligodeoxynucleotides (ODNs). The hybridization ability of the obtained ODN derivatives containing 2 with complementary strands was evaluated by melting temperature (T(m)) measurements. As a result, the ODN derivatives hybridized with DNA and RNA complements in a sequence-selective manner, though the stability of the duplexes was lower than that of the corresponding natural DNA/DNA or DNA/RNA duplex.     

Highly sequence specific RNA terminal labeling by DNA photoligation

We report the nonenzymatic terminal labeling of oligoribonucleotide (ORN) by using template-directed photoligation through 5-carboxyvinyl-2'-deoxyuridine ((CV)U) with high selectivity.     

杂交及切断过程的QCM实时检测研究(英文)

采用自组装技术,将 5′端标记有巯基的 20-merODN(oligo 1)以金 硫键形式牢固结合在 7. 995MHz的AT-切石英晶体的镀金表面,然后由石英晶体微天平实时检测了与碱基序列互补的 10 merODN (oligo 2)和 8 merODN(oligo 3)的杂交,同时还研究了稀土金属铈离子在温和条件下对DNA的水解切断作用.结果表明:应用QCM方法可能实时检测DNA的固定和杂交,Ce(IV)能随机切断单链DNA;但不能切断杂交形成的双链DNA,因此可利用杂交保护的方法对单链DNA实行定位切断.     

Efficient C2'alpha-hydroxylation of deoxyribose in protein-induced Z-form DNA

DNA local conformations are thought to play an important biological role in processes such as gene expression by altering DNA-protein interactions. Although left-handed Z-form DNA is one of the best-characterized and significant local structures of DNA, having been extensively investigated for more than two decades, the biological relevance of Z-form DNA remains unclear. This is presumably due to the lack of a versatile detection method in a living cell. Previously, we demonstrated that the incorporation of a methyl group at the guanine C8 position (m(8)G) dramatically stabilizes the Z-form of short oligonucleotides in a variety of sequences. To develop a photochemical method to detect Z-form DNA, we examined the photoreaction of 5-iodouracil-containing Z-form d(CGCG(I)UGCG)(ODN 1)/d(Cm(8)GCAm(8)GCG)(ODN 2) in 2 M NaCl and found stereospecific C2'alpha-hydroxylation occurred at G(4) to provide d(CGCrGUGCG), 5. Recently, Rich and co-workers [Schwartz et al. Science 1999, 284, 1841. Schwartz et al. Nat. Struct. Biol. 2001, 8, 761] found that an ubiquitous RNA editing enzyme, adenosine deaminase 1 (ADAR1), and tumor-associated protein DML-1 specifically bind to Z-form DNA. In the present study, we investigate the photoreactivity of octanucleotide ODN 1-2 in Z-form induced by Zalpha, which is the NH(2)-terminal domain of ADAR1 responsible for tight binding of ADAR1. Detailed product analysis revealed that the C2'alpha-hydroxylated products 5 and 6 produced significantly higher yields in Z-form ODN 1-2 induced by Zalpha compared with that in 2 M NaCl. Upon treatment with ribonuclease T1, 5 and 6 were quantitatively hydrolyzed at the 3'-phosphodiester bond of the rG residue to provide d(UGCG) as a common hydrolyzed fragment on the 3' side. Quantitative analysis demonstrated that the amount of photochemically formed 5 and 6 from ODN 1-2 directly correlated with the proportion of Z-form induced by Zalpha or NaCl. These results suggest that this photochemical and enzymatic procedure can be used as a specific probe for the existence of local Z-form structure in cellular DNA.     

Synthesis and characterization of a [3-15N]-labeled cis-syn thymine dimer-containing DNA duplex

Cis-syn thymine dimers are the major photoproducts of DNA and are the principal cause of mutations induced by sunlight. To better understand the nature of base pairing with cis-syn thymine dimers, we have synthesized a decamer oligodeoxynucleotide (ODN) containing a cis-syn thymine dimer labeled at the N3 of both T's with 15N by two efficient routes from [3-15N]-thymidine phosphoramidite. In the postsynthetic irradiation route, an ODN containing an adjacent pair of [3-15N]-labeled T's was irradiated and the cis-syn dimer-containing ODN isolated by HPLC. In the mixed building block route, a mixture of cis-syn and trans-syn dimer-containing ODNs was synthesized from a mixture of [3-15N]-labeled thymine dimer phosphoramidites after which the cis-syn dimer-containing ODN was isolated by HPLC. The N3-nitrogen and imino proton signals of an (15)N-labeled thymine dimer-containing decamer duplex were assigned by 2D 1H-15N heterocorrelated HSQC NMR spectroscopy, and the 15N-1H coupling constant was found to be 1.8 Hz greater for the 5'-T than for the 3'-T. The larger coupling constant is indicative of weaker H-bonding that is consistent with the more distorted nature of the 5'-base pair found in solution state NMR and crystallographic structures.     

Photosensitization of DNA damage by a new cationic pyropheophorbide derivative: sequence-specific formation of a frank scission

Pyropheophorbides are red-absorbing porphyrin-like photosensitizers that may interact with DNA either by intercalation or by external binding with self-stacking according to the value of the nucleotide to chromophore molar ratio (N/C). This article reports on the nature and sequence selectivity of the DNA damage photoinduced by a water-soluble chlorhydrate of aminopyropheophorbide. First, this pyropheophorbide is shown to induce on irradiation the cleavage of phiX174 DNA by both Type-I and -II mechanisms, suggested by scavengers and D2O effects. These conclusions are then improved by sequencing experiments performed on a 20-mer oligodeoxynucleotide (ODN) irradiated at wavelengths >345 nm in the presence of the dye, N/C varying from 2.5 to 0.5. Oxidation of all guanine residues to the same extent is observed after piperidine treatment on both single- and double-stranded ODN. Moreover, unexpectedly, a remarkable sequence-selective cleavage occurring at a 5'-CG-3' site is detected before alkali treatment. This frank break is clearly predominant for a low nucleotide to chromophore molar ratio, corresponding to a self-stacking of the dye along the DNA helix. The electrophoretic properties of the band suggest that this lesion results from a sugar oxidation, which leads via a base release to a ribonolactone residue. The proposal is supported by high-performance liquid chromatography-matrix-assisted laser desorption-ionization mass spectrometry experiments that also reveal other sequence-selective frank scissions of lower intensity at 5'-GC-3' or other 5'-CG-3' sites. This sequence selectivity is discussed with regard to the binding selectivity of cationic porphyrins.     

Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.     

NMR studies of DNA single strands and DNA:RNA hybrids with and without 1-propynylation at C5 of oligopyrimidines

The 1-propynylation at C5 of consecutive pyrimidines in DNA can enhance DNA:RNA hybrid stability at 37 degrees C by over 1 kcal/mol of substitution [Barnes, T. W., III; Turner, D. H. J. Am. Chem. Soc.2001, 123, 4107-4118]. To provide information on the structural consequences of propynylation, two-dimensional NMR spectroscopy was used to study the structures of several oligonucleotides. Intraresidue nuclear Overhauser effect spectroscopy cross peaks were observed at 30 degrees C and a 200 ms mixing time in the H6-H1' region for 5'(dC(P)C(P)U(P)C(P)C(P)U(P)U(P)) (ssPrODN) but not for 5'(dCCUCCUU) (ssODN), suggesting preorganization of the propynylated single strand. NMR structures of the duplexes 5'(dC(P)C(P)U(P)C(P)C(P)U(P)U(P))3':3'(rGAGGAGGAAAU)5' (PrODN:RNA), 5'(dCC(P)U(P)C(P)C(P)U(P)U(P))3':3'(rGAGGAGGAAAU)5' (sPrODN1:RNA), and 5'(dCCUCCUU)3':3'(rGAGGAGGAAAU)5' (ODN:RNA) indicate that their global structures are almost identical. The NMR data, however, suggest that the 5'-end of sPrODN1:RNA is more dynamic than that of PrODN:RNA. In the propynylated duplexes, the propyne group stacks on the aromatic ring of the 5'-base and extends into the major groove. The results suggest that the increased stability of the propynylated duplexes is caused by preorganization of the propynylated single strand and different interactions in the double strand. The propynyl group provides volume exclusion, enhanced stacking, and possibly different solvation.     

Highly efficient photochemical 2'-deoxyribonolactone formation at the diagonal loop of a 5-iodouracil-containing antiparallel G-quartet

To explore the structure-dependent hydrogen abstraction in antiparallel and parallel G-quartet DNA structures, the photochemical reactivity of 5-iodouracil ((I)U)-containing human telomeric DNA 22-mers was investigated under the 302 nm UV irradiation conditions. We discovered that only antiparallel ODN 4, in which the second T residue in the diagonal loop of the antiparallel G-quartet is substituted with (I)U, was rapidly consumed as compared with parallel ODN 4 and the other (I)U-containing 22-mers under the irradiation conditions. Product analysis of the photolyzate of antiparallel ODN 4 indicated that a 2'-deoxyribonolactone residue was effectively produced at the 5' side of the (I)U residue in the diagonal loop. Photochemical 2'-deoxyribonolactone formation was also found in the (I)U-containing diagonal loop of antiparallel G-quartets d(GGGGTTT(I)UGGGG)(2) and d(GGGGTT(I)UTGGGG)(2), whereas the reaction did not occur at other DNA structures, including the single-stranded form, the loop region of the hairpin, and linear four-stranded G-quartets. The results clearly indicate that this type of 2'-deoxyribonolactone formation efficiently occurrs only in the diagonal loop of the antiparallel G-quartet. Furthermore, we found that 2'-deoxyribonolactone was formed at the (I)U-containing G-rich sequence of the IgG switch regions and the 5' termini of the Rb gene, suggesting the formation of an antiparallel G-quartet with a diagonal loop in these sequences. These results suggest that the present photochemical method can be used as a specific probe for the antiparallel G-quartet with the diagonal loop.     

Syntheses of 5-formyl- and 5-carboxyl-dC containing DNA oligos as potential oxidation products of 5-hydroxymethylcytosine in DNA

To investigate the potential oxidation products of 5-hydroxymethylcytosine (5-hmC)-containing DNA, we present here efficient syntheses of 5-formyl- and 5-methoxycarbonyl-2'-deoxycytidine phosphoramidites. The 5-formyl group in III was easy to introduce and was compatible with phosphoramidite and DNA syntheses. An additional treatment of ODN1 with NaBH(4) produced the corresponding ODN2 quantitatively. Phosphoramidite V was also incorporated into DNA, and the methyl ester could be hydrolyzed under mild basic conditions to afford ODN3.