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Complementary Strand (complementary + strand)

Distribution by Scientific Domains

Chemistry	92%

Selected Abstracts

An Aptamer-Based Bound/Free Separation System for Protein Detection

ELECTROANALYSIS, Issue 11 2009
Mieko Fukasawa
Abstract Aptamer hybridizes with its complementary strand. However, the complementary strand has difficulties to hybridize with the aptamer bound to a target because the aptamer forms higher-order structures. Exploiting this property, we developed simple bound/free separation systems for thrombin and IgE detection. The complementary strand was immobilized onto beads and the aptamer was labeled with pyrroquinoline quinone glucose dehydrogenase (PQQGDH). In the absence of a target, the aptamer is trapped by beads, whereas in the presence of a target, the aptamer bound to the target is not trapped. Thus the aptamer-target complexes can be recovered easily and detected by PQQGDH activity. This system allow the detection of 270,pM thrombin and 1,nM IgE. [source]

Oligonucleotide Duplexes with Tethered Photoreactive Ruthenium(II) Complexes: Influence of the Ligands and Their Linker on the Photoinduced Electron Transfer and Crosslinking Processes of the Two Strands

EUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 4 2009
Stéphanie Deroo
Abstract The photoreactivity of new RuII -oligonucleotide conjugates is investigated in the presence of their complementary strands. The goal is to determine the origins of different effects of parameters that control the photocrosslinking process of the two strands. Therefore, two RuII compounds, either [Ru(tap)3]2+or [Ru(tap)2phen]2+ (tap = 1,4,5,8-tetraazaphenanthrene, phen = 1,10-phenanthroline) with different oxidation powers, were tethered with different linkers to either the 5,- or 3,-phosphate end of the probe strand before hybridization with the complementary strand. These systems were studied by time-resolved emission spectroscopy, UV/Vis absorption experiments, PAGE and MS (ESI) analyses. The best yields of photocrosslinking (45,%) obtained with [Ru(tap)3]2+ tethered to the 3,-position are due to (i) a higher oxidation power of the complex and (ii) its attachment at the 3,-position. Indeed, this tethering favours the interaction of the Ru compound with the duplex and, therefore, inhibits its photodechelation. This work allows better design of sequence-specific DNA photodamaging agents prior to biological applications.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source]

Molecular dynamics simulation of clustered DNA damage sites containing 8-oxoguanine and abasic site

JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 8 2005
Hirofumi Fujimoto
Abstract Clustered DNA damage sites induced by ionizing radiation have been suggested to have serious consequences to organisms, such as cancer, due to their reduced probability to be repaired by the enzymatic repair machinery of the cell. Although experimental results have revealed that clustered DNA damage sites effectively retard the efficient function of repair enzymes, it remains unclear as to what particular factors influence this retardation. In this study, approaches based on molecular dynamics (MD) simulation have been applied to examine conformational changes and energetic properties of DNA molecules containing clustered damage sites consisting of two lesioned sites, namely 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic (AP) site, located within a few base pairs of each other. After 1 ns of MD simulation, one of the six DNA molecules containing a clustered damage site develops specific characteristic features: sharp bending at the lesioned site and weakening or complete loss of electrostatic interaction energy between 8-oxoG and bases located on the complementary strand. From these results it is suggested that these changes would make it difficult for the repair enzyme to bind to the lesions within the clustered damage site and thereby result in a reduction of its repair capacity. © 2005 Wiley Periodicals, Inc. J Comput Chem 26: 788,798, 2005 [source]

The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide-oligonucleotide adducts determined using nanoLC/MS/MS

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 8 2009
James Glick
Abstract Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, ( ± )-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [( ± )- anti -benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5,-ACCCG5CG7TCCG11CG13C-3,/5,-GCGCGGGCGCGGGT-3,) derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational ,hot spots' and have also been reported as chemical ,hot spots' for the formation of BPDE-DNA adducts. In evaluating the effect of cytosine methylation on BPDE-DNA adduct binding, it was found that codon 156, containing the nucleobase G5 instead of the mutational hot spot codons 157 (G7) and 158 (G11), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G5, G11 > G13 > G7. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G11 CpG site (codon 158) approximately two-fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G5, G7 > G11 > G13. The three- to four-fold increase in adduction at the mutational hot spot codon 157 (G7) relative to the unmethylated or single-stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Real-time Detection of Nucleotide Incorporation During Complementary DNA Strand Synthesis

CHEMBIOCHEM, Issue 7 2003
Alexander Krieg
Abstract Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction. [source]

Photocrosslinking in Ruthenium-Labelled Duplex Oligonucleotides

CHEMBIOCHEM, Issue 2-3 2003
O. Lentzen
Abstract The formation of a photoadduct between a [Ru(1,4,5,8-tetraazaphenanthrene)24,7-diphenylphenanthroline]2+complex chemically attached to a synthetic oligonucleotide, and a guanine moiety in a complementary targeted single-stranded DNA molecule was studied for ten 17-mer duplexes by denaturing gel electrophoresis. This photoadduct formation leads to photocrosslinking of the two strands. The percentage quenching of luminescence of the complex by electron transfer was compared to the resulting yield of photocrosslinked product. This yield does not only depend on the ionisation potential of the guanine bases, which are electron donors, but also on other factors, such as the position of the guanine bases as compared to the site of attachment of the complex. The photocrosslinking yield is higher when the guanine moieties are towards the 3, end on the complementary strand as compared to the tethering site. Computer modelling results are in agreement with this preference for the 3, side for the photoreaction. Interestingly, the photocrosslink is not alkali labile. Moreover, a type III exonuclease enzyme is blocked at the position of photocrosslinking. [source]

Solution Structure and Stability of Tryptophan-Containing Nucleopeptide Duplexes

CHEMBIOCHEM, Issue 1 2003
Irene Gómez-Pinto
Abstract Covalently linked peptide,oligonucleotide hybrids were used as models for studying tryptophan,DNA interactions. The structure and stability of several hybrids in which peptides and oligonucleotides are linked through a phosphodiester bond between the hydroxy group of a homoserine (Hse) side chain and the 3,-end of the oligonucleotide, have been studied by both NMR and CD spectroscopy and by restrained molecular dynamics methods. The three-dimensional solution structure of the complex between Ac-Lys-Trp-Lys-Hse(p3,dGCATCG)-Ala-OH (p=phosphate, Ac=acetyl) and its complementary strand 5,dCGTAGC has been determined from a set of 276 experimental NOE distances and 33 dihedral angle constraints. The oligonucleotide structure is a well-defined duplex that belongs to the B-form family of DNA structures. The covalently linked peptide adopts a folded structure in which the tryptophan side chain stacks against the 3,-terminal guanine moiety, which forms a cap at the end of the duplex. This stacking interaction, which resembles other tryptophan,nucleobase interactions observed in some protein,DNA complexes, is not observed in the single-stranded form of Ac-Lys-Trp-Lys-Hse(p3,dGCATCG)-Ala-OH, where the peptide chain is completely disordered. A comparison with the pure DNA duplex, d(5,GCTACG3,),(5,CGTAGC3,), indicates that the interaction between the peptide and the DNA contributes to the stability of the nucleopeptide duplex. The different contributions that stabilize this complex have been evaluated by studying other nucleopeptide compounds with related sequences. [source]

Hybridization-Sensitive On,Off DNA Probe: Application of the Exciton Coupling Effect to Effective Fluorescence Quenching

CHEMISTRY - AN ASIAN JOURNAL, Issue 6 2008
Shuji Ikeda Dr.
Abstract The design of dyes that emit fluorescence only when they recognize the target molecule, that is, chemistry for the effective quenching of free dyes, must play a significant role in the development of the next generation of functional fluorescent dyes. On the basis of this concept, we designed a doubly fluorescence-labeled nucleoside. Two thiazole orange dyes were covalently linked to a single nucleotide in a DNA probe. An absorption band at approximately 480,nm appeared strongly when the probe was in a single-stranded state, whereas an absorption band at approximately 510,nm became predominant when the probe was hybridized with the complementary strand. The shift in the absorption bands shows the existence of an excitonic interaction caused by the formation of an H aggregate between dyes, and as a result, emission from the probe before hybridization was suppressed. Dissociation of aggregates by hybridization with the complementary strand resulted in the disruption of the excitonic interaction and strong emission from the hybrid. This clear change in fluorescence intensity that is dependent on hybridization is useful for visible gene analysis. [source]

A Cryptophane Biosensor for the Detection of Specific Nucleotide Targets through Xenon NMR Spectroscopy

CHEMPHYSCHEM, Issue 14 2007
Vincent Roy Dr.
DNA sensor: A xenon host composed of a cryptophane structure with a DNA strand (see picture) serves to detect its complementary strand in the micromolar range through laser-polarized 129Xe NMR spectroscopy. [source]

Gel immobilization of acrylamide-modified single-stranded DNA template for pyrosequencing

ELECTROPHORESIS, Issue 12 2007
Pengfeng Xiao Dr.
Abstract A novel two-step process was developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1,M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electrophoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate (PPi), and excess deoxyribonucleotides under alkali conditions. The results show that the 3-D polyacrylamide gel network has a high immobilization capacity and the modified PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10,,L of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the crude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be incorporated into a DNA-chip format for high-throughput pyrosequencing analysis. [source]

Oligonucleotide Duplexes with Tethered Photoreactive Ruthenium(II) Complexes: Influence of the Ligands and Their Linker on the Photoinduced Electron Transfer and Crosslinking Processes of the Two Strands

Discrimination of G-Quadruplexes from Duplex and Single-Stranded DNAs with Fluorescence and Energy-Transfer Fluorescence Spectra of Crystal Violet

CHEMISTRY - A EUROPEAN JOURNAL, Issue 4 2009
De-Ming Kong Dr.
Abstract G-rich nucleic acid sequences with the potential to form G-quadruplex structures are common in biologically important regions. Most of these sequences are present with their complementary strands, so the development of a sensitive biosensor to distinguish G-quadruplex and duplex structures and to determine the competitive ability of quadruplex to duplex structures has received a great deal of attention. In this work, the interactions between two triphenylmethane dyes (malachite green (MG) and crystal violet (CV)) and G-quadruplex, duplex, or single-stranded DNAs were studied by fluorescence spectroscopy and energy-transfer fluorescence spectroscopy. Good discrimination between quadruplexes and duplex or single-stranded DNAs can be achieved by using the fluorescence spectrum of CV or the energy-transfer fluorescence spectra of CV and MG. In addition, by using energy-transfer fluorescence titrations of CV with G-quadruplexes, the binding-stoichiometry ratios of CV to G-quadruplexes can be determined. By using the fluorescence titrations of G-quadruplex,CV complexes with C-rich complementary strands, the fraction of G-rich oligonucleotide that engages in G-quadruplex structures in the presence of the complementary sequence can be measured. This study may provide a simple method for discrimination between quadruplexes and duplex or single-stranded DNAs and for measuring G-quadruplex percentages in the presence of the complementary C-rich sequences. [source]

Highly Fluorescent Conjugated Pyrenes in Nucleic Acid Probes: (Phenylethynyl)pyrenecarbonyl-Functionalized Locked Nucleic Acids

CHEMISTRY - A EUROPEAN JOURNAL, Issue 35 2008
Irina
Abstract In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M1,M6 and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M1,M6 in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl,LNA monomers M4, M5, and M6 proved highly useful for the detection of single mismatches in DNA/RNA targets. [source]