RESUMO
To clarify the cis-trans isomeric effect on the ultrafast DNA photo-cross-linking reaction of 3-cyanovinylcarbazole nucleoside ((CNV)K) in the DNA duplex, which gives a single photodimer on the reversed-phase HPLC chromatogram, the kinetics of the cis-trans photoisomerization of (CNV)K in double-stranded DNA was evaluated. Since the photoisomerization rate constant for cis to trans isomerization in double-stranded DNA was significantly larger than that for trans to cis isomerization, and the thermodynamic stability of the trans isomer was higher than that of the cis isomer, it was strongly suggested that the trans isomer of (CNV)K is a reactive species of the photo-cross-linking reaction. (1)H-(1)H NOESY analysis of the photoadduct consisting of (CNV)K and T also supported the trans-mediated photo-cross-linking reaction of (CNV)K. By using this ultrafast photo-cross-linking reaction for the molecular beacon-based SNPs typing, four individual Japanese rice strains were clearly distinguishable with simple photoirradiation and fluorescence imaging using double-stranded target DNAs.
Assuntos
Carbazóis/química , Reagentes de Ligações Cruzadas/química , DNA/química , DNA/genética , Genótipo , Nucleosídeos/química , Indicadores e Reagentes , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Estereoisomerismo , TermodinâmicaRESUMO
3-Cyanovinylcarbazole nucleoside, which effectively photocrosslinks to the pyrimidine base in complementary RNA strands, was incorporated into antisense oligonucleotides, and we evaluated the photoreactivity and the sequence selectivity to mutated K-ras oligoRNAs, as well as the regulation of the function of K-ras mRNA. We demonstrated that the reverse transcription and the translation activity of K-ras mRNA were quickly suppressed by a few seconds of photoirradiation with the addition of the photoresponsive antisense ODN.
Assuntos
RNA Mensageiro/metabolismo , Raios Ultravioleta , Carbazóis/química , Reagentes de Ligações Cruzadas/química , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/genética , Processos Fotoquímicos , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas/química , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais CultivadasRESUMO
We present a new type of nanoparticle-based DNA sensor using surface-enhanced Raman scattering (SERS) on gold nanoparticle (Au NP) aggregates formed by DNA photoligation. The DNA sensor exploits the photoligation reaction between oligodeoxynucleotides (ODNs) attached to the surfaces of Au NPs in the presence of target DNA (T-DNA). When hybridization takes place between the ODNs and T-DNA, Au NPs are covalently crosslinked to form aggregates via photoligation. Once the NP aggregates are formed, the interspace between Au NPs in the aggregate act as a stable "hot spot", and a SERS signal from the Raman-active molecules (sodium cacodylate) present in the hot spot is easily and sensitively detected. In contrast, a SERS signal is not detected if the hybridization is unsuccessful, because the stable hot spot does not form. This DNA sensor does not require an enzymatic reaction, fluorescent dye, precise temperature control, or complicated operating procedures.
Assuntos
DNA/química , Ouro/química , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Ácido Cacodílico/química , DNA/análise , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Processos Fotoquímicos , Espectrofotometria UltravioletaRESUMO
We describe a photoreversible DNA end capping via 3-cyanovinylcarbazole nucleoside. Doubly end-capped oligodeoxynucleotide (ODN) exhibits increased stability against snake venom phosphodiesterase and shows high thermal stability.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , DNA/metabolismo , Oligodesoxirribonucleotídeos/química , Reagentes de Ligações Cruzadas/metabolismo , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Fosfodiesterase I/metabolismo , Fotoquímica , Venenos de Serpentes/metabolismo , TemperaturaRESUMO
We describe a simple and inexpensive single-nucleotide polymorphism (SNP) typing method, using DNA photoligation with 5-carboxyvinyl-2'-deoxyuridine and two fluorophores. This SNP-typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity up to 10 000. This method can be used in the SNP typing of actual genomic DNA samples from food crops.
RESUMO
We describe a novel ultrafast reversible DNA interstrand photo-cross-linking reaction via 3-cyanovinylcarbazole nucleoside ( (CNV)K). Oligodeoxynucleotide (ODN) containing (CNV)K can be photo-cross-linked by irradiation at 366 nm for 1 s, and the photo-cross-linked ODN can be split by irradiation at 312 nm for 60 s.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , DNA/efeitos da radiação , Carbazóis/química , Carbazóis/efeitos da radiação , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas/efeitos da radiação , Nitrilas/química , Nitrilas/efeitos da radiação , Nucleosídeos/química , Nucleosídeos/efeitos da radiação , Oligonucleotídeos/química , Oligonucleotídeos/efeitos da radiação , Fotoquímica , Compostos de Vinila/química , Compostos de Vinila/efeitos da radiaçãoRESUMO
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.
Assuntos
Carbazóis/química , DNA/efeitos da radiação , Oligodesoxirribonucleotídeos/efeitos da radiação , Uracila/análogos & derivados , Uracila/química , Sequência de Bases , Luz , Estrutura MolecularRESUMO
We synthesized a novel fluorescent analogue of acetylcholine, Cy3-3-acylcholine. The molecular weight of the products agreed with structural predictions. Discrete intensity changes of fluorescent spots due to a single ligand binding/unbinding to nAChR were visualized by TIRF microscopy. The agonist effect of the Cy3-3-acylcholine on nicotinic acetylcholine receptor (nAChR) was confirmed electrophysiologically. This newly synthesized fluorescent analogue will enable us to conduct more elaborate studies on single channel interaction processes between nAChR and ligands.
Assuntos
Acetilcolina/análogos & derivados , Canais Iônicos/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/síntese química , Acetilcolina/química , Animais , Atropina/farmacologia , Colinérgicos , Fluorescência , Modelos Biológicos , Estrutura Molecular , Oócitos , XenopusRESUMO
We describe a highly efficient method for reversible photocircularization of oligonucleotide (ODN) on a double-stranded DNA template: 5-carboxyvinyl-2'-deoxyuridine-containing ODN was reversibly circularized around the target sequence of the double-stranded plasmid DNA resulting in formation of a catenated plasmid.
Assuntos
DNA/química , Nucleotídeos/química , Sequência de Bases , DNA/efeitos da radiação , DNA Circular/química , DNA Circular/efeitos da radiação , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos da radiação , Fotoquímica , Plasmídeos/química , Raios UltravioletaRESUMO
We describe a highly efficient template-directed photoligation of oligodeoxynucleotides (ODNs) through 5-carbamoylvinyl-2'-deoxycytidine ((CV)C). When an ODN containing (CV)C at the 5' end was photoirradiated with an ODN containing a pyrimidine base at the 3' end in the presence of template DNA, efficient photoligation was observed without any byproduct formation. Single nucleotide differences can be successfully distinguished by using photoligation-based DNA chip assay. [structure: see text]
Assuntos
DNA/química , Desoxicitidina/química , Oligonucleotídeos/química , Sequência de Bases , Desoxicitidina/análogos & derivados , Fluorescência , Estrutura Molecular , FotoquímicaRESUMO
We report that deamination coupled with 5-carboxyvinyldeoxyuridine-mediated photobranching causes the heat-induced transition of cytosine to uracil with high efficiency without any side reaction.
Assuntos
Citosina/química , DNA/química , Uracila/química , Sequência de Bases , DNA/genética , Dados de Sequência Molecular , Mutação/genética , FotoquímicaAssuntos
Citosina/química , DNA/química , Mutagênese Sítio-Dirigida/métodos , Oligodesoxirribonucleotídeos/química , Uracila/química , Citosina/metabolismo , DNA/genética , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Processos Fotoquímicos , Uracila/metabolismoRESUMO
[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.
Assuntos
DNA/química , DNA/efeitos da radiação , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Oligodesoxirribonucleotídeos/síntese química , Sequência de Bases , Estrutura Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Fotoquímica , Moldes GenéticosRESUMO
A modified oligodeoxynucleotide (ODN) containing N(3)-methyl-5-cyanovinyl-2'-deoxyuridine reacts by photoirradiation at 366 nm with an adenine residue of a complementary template ODN to yield an end-capped ODN in 87% yield.
Assuntos
DNA/química , Desoxiuridina/análogos & derivados , Sequência de Bases , Desoxiuridina/química , FotoquímicaRESUMO
To develop a covalent and specific labeling method for single- and double-stranded plasmid DNA, photoresponsive oligonucleotide containing 3-cyanovinylcarbazole nucleoside was adopted. Single- and double-stranded plasmid DNA was successfully labeled/de-labeled with Cy3 and/or biotin by photoirradiation.
Assuntos
DNA/química , Oligonucleotídeos/química , Plasmídeos/genética , Coloração e Rotulagem/métodos , Acrilonitrila/análogos & derivados , Acrilonitrila/química , Carbazóis/química , Corantes Fluorescentes/química , Processos FotoquímicosRESUMO
Photo-induced artificial RNA editing was demonstrated using photo-reactive oligonucleotides containing 3-cyanovinylcarbazole nucleoside. This non-enzymatic and sequence-specific methodology will make a major contribution to the elucidation of RNA functions including non-coding RNAs and to the development of drugs based on sequence-specific RNA editing.
Assuntos
Citosina/química , Oligonucleotídeos/química , Fotoquímica/métodos , Edição de RNA , RNA/química , Uracila/química , Sequência de Bases , Carbazóis/química , Nucleosídeos/químicaRESUMO
We describe a novel reversible interstrand photocrosslinking reaction of RNA. In this system, a modified oligodeoxynucleotide (ODN) containing 3-cyanovinylcarbazole nucleoside ((CNV)K) reacts by photoirradiation at 366 nm for 1 s with pyrimidine residue of a complementary template RNA to yield a crosslinked product in 94% yield.
Assuntos
RNA/química , Carbazóis/química , DNA/química , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/efeitos da radiação , Processos Fotoquímicos , RNA/efeitos da radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We describe a selective and sensitive single-nucleotide polymorphism (SNP) typing of genes from rice cultivars by using DNA photoligation. This SNP typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity.
Assuntos
Genes de Plantas , Oryza/genética , Polimorfismo de Nucleotídeo Único , DNA de Plantas/química , DNA de Plantas/efeitos da radiação , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Análise de Sequência de DNARESUMO
The synthesis of a photoresponsive alpha-5-cyanovinyl- 2',3'-dideoxyuridine-5'-triphosphate (alpha dd(C)UTP) is described. A one-pot enzymatic synthesis of photoresponsive alpha-5-cyanovinyl-2',3'-dideoxyuridine (alpha dd(C)U) labeled ODNs with terminal deoxynucleotideyl transferase (TdT) would be a powerful tool for the development of modified long-chain DNA and branched DNA unable to be prepared by DNA synthesizer or enzymatic ligation.