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2-Aminopurine-Modified Abasic-Site-Containing Duplex DNA for Highly Selective Detection of Theophylline

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.
Journal of the American Chemical Society (Impact Factor: 11.44). 03/2009; 131(7):2448-9. DOI: 10.1021/ja8095625
Source: PubMed

ABSTRACT 2-Aminopurine-modified abasic-site-containing duplex [DNA 5'-TCTGC GTCCT PXT TAACG CACAC-3'/3'-AGACG CAGGA TCA ATTGC GTGTG-5'; P = 2-aminopurine, X = abasic site (Spacer-C3), C = receptor base] is capable of selectively binding to the bronchodilator theophylline with a dissociation constant of 10 microM (5 degrees C, pH 7.0, I = 0.11 M) and is applicable to monitoring serum theophylline concentrations.

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    • "Fluorescence life time assay has been proved to be a useful tool to study the changes in local base stacking interactions in DNA, one particularly interesting report being the effect of metal ions and sequence on abasic sites [31]. Relevant studies conducted so far using this analytical tool often involves the substitution of a DNA base with a modified base that act as a fluorescent probe [44] [45] [46]. In a novel intervention, we are reporting the efficiency of APE1 induced cleavage at clustered abasic sites as evaluated by time resolved and steady state fluorescence spectroscopy, without the use of any modified nucleotide fluorescent probe. "
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    ABSTRACT: Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of Km was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks. Copyright © 2014 Elsevier B.V. All rights reserved.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2014; 766767:56-65. DOI:10.1016/j.mrfmmm.2014.06.002 · 4.44 Impact Factor
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    • "Fluorescence life time assay has been proved to be a useful tool to study the changes in local base stacking interactions in DNA, one particularly interesting report being the effect of metal ions and sequence on abasic sites [31]. Relevant studies conducted so far using this analytical tool often involves the substitution of a DNA base with a modified base that act as a fluorescent probe [44] [45] [46]. In a novel intervention, we are reporting the efficiency of APE1 induced cleavage at clustered abasic sites as evaluated by time resolved and steady state fluorescence spectroscopy, without the use of any modified nucleotide fluorescent probe. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of Km was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks.
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    • "Such a blocking oligonucleotide must be specifically designed to target predator DNA and thus bind preferentially with predator sequences, limiting their amplification. This concept has been effectively used in the field of clinical chemistry (Kageyama et al. 2008; Wang et al. 2008; Li et al. 2009) and in environmental microbiology (Liles et al. 2003). However, the application of blocking oligonucleotide in trophic studies is relatively recent. "
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    ABSTRACT: Diet analysis is a prerequisite to fully understand the biology of a species and the functioning of ecosystems. For carnivores, traditional diet analyses mostly rely upon the morphological identification of undigested remains in the faeces. Here, we developed a methodology for carnivore diet analyses based on the next-generation sequencing. We applied this approach to the analysis of the vertebrate component of leopard cat diet in two ecologically distinct regions in northern Pakistan. Despite being a relatively common species with a wide distribution in Asia, little is known about this elusive predator. We analysed a total of 38 leopard cat faeces. After a classical DNA extraction, the DNA extracts were amplified using primers for vertebrates targeting about 100 bp of the mitochondrial 12S rRNA gene, with and without a blocking oligonucleotide specific to the predator sequence. The amplification products were then sequenced on a next-generation sequencer. We identified a total of 18 prey taxa, including eight mammals, eight birds, one amphibian and one fish. In general, our results confirmed that the leopard cat has a very eclectic diet and feeds mainly on rodents and particularly on the Muridae family. The DNA-based approach we propose here represents a valuable complement to current conventional methods. It can be applied to other carnivore species with only a slight adjustment relating to the design of the blocking oligonucleotide. It is robust and simple to implement and allows the possibility of very large-scale analyses.
    Molecular Ecology 01/2012; 21(8):1951-65. DOI:10.1111/j.1365-294X.2011.05424.x · 6.49 Impact Factor
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