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Michael J. Waring - One of the best experts on this subject based on the ideXlab platform.
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Articles Sequence-Selective Interaction of the Minor-Groove Interstrand Cross-Linking Agent SJG-136 with Naked and Cellular DNA: Footprinting and Enzyme Inhibition
2020Co-Authors: C Martin, Tom Ellis, Claire J. Mcgurk, Terence C. Jenkins, Ja Hartley, Michael J. Waring, David Edwin ThurstonAbstract:SJG-136 (3) is a novel pyrrolobenzodiazepine (PBD) dimer that is predicted from molecular models to bind in the minor groove of DNA and to form sequence-selective interstrand cross-links at 5'-Pu-GATC-Py-3' (Pu ) purine; Py ) pyrimidine) sites through covalent bonding between each PBD unit and guanines on opposing strands. Footprinting studies have confirmed that high-affinity adducts do form at 5'-G-GATC-C-3' sequences and that these can inhibit RNA polymerase in a sequence-selective manner. At higher concentrations of SJG-136, bands that migrate more slowly than one of the 5'-G-GATC-C-3' footprint sites show significantly reduced intensity, concomitant with the appearance of higher molecular weight material near the gel origin. This phenomenon is attributed to interstrand cross-linking at the 5 '-G- GATC-C-3' site and is the first report of DNA Footprinting being used to detect interstrand cross-linked adducts. The control dimer GD113 (4), of similar structure to SJG-136 but unable to cross-link DNA due to its C7/C7'-linkage rather than C8/C8'-linkage, neither produces footprints with the same DNA sequence nor blocks transcription at comparable concentrations. In addition to the two high-affinity 5 '-G-GATC-C-3' footprints on the MS2 DNA sequence, other SJG-136 adducts of lower affinity are observed that can still block transcription but with lower efficiency. All these sites contain the 5'-GXXC-3' motif (where XX includes AG, TA, GC, CT, TT, GG, and TC) and represent less-favored cross-link sites. In time-course experiments, SJG-136 blocks transcription if incubated with a double-stranded DNA template before the transcription components are added; addition after transcription is initiated fails to elicit blockage. Single-strand ligation PCR studies on a sequence from the c-jun gene show that SJG-136 binds to 5'-GAAC-3'/5'-GTTC-3' (preferred) or 5'- GAGC-3'/5'-GCTC-3' sequences. Significantly, adducts are obtained at the same sequences following extraction of DNA from drug-treated K562 cells, confirming that the agent reaches the cellular genome and interacts with the DNA in a sequence-selective fashion. Finally, SJG-136 efficiently inhibits the action of restriction endonuclease BglII, which has a 5'-A-GATC-T-3' motif at its cleavage site.
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visualising DNA Footprinting and 1 2d gels
Molecular BioSystems, 2005Co-Authors: Adam R Urbach, Michael J. WaringAbstract:The study of molecular recognition of DNA by natural and synthetic ligands has made enormous progress due in large part to the discovery and development of methods for separating DNA fragments by gel electrophoresis in one and two dimensions, and for characterizing DNA–ligand complexes by Footprinting techniques.
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2,3-Bifunctionalized Quinoxalines: Synthesis, DNA Interactions and Evaluation of Anticancer, Anti-tuberculosis and Antifungal Activity
Molecules, 2002Co-Authors: Michael J. Waring, Taibi Ben-hadda, Ann T. Kotchevar, A. Ramdani, Rachid Touzani, Sghir Elkadiri, Abdelkader Hakkou, Mohamed Bouakka, Tom EllisAbstract:A variety of 2,3-bifunctionalized quinoxalines (6-14) have been prepared by the condensation of 1,6-disubstituted-hexan-1,3,4,6-tetraones (1-4) with o-phenylenediamine, (R,R)-1,2-diaminocyclohexane and p-nitro-o-phenylenediamine. It is concluded that strong intramolecular N-H----O bonds in the favoured keto-enamine form may be responsible for the minimal biological activities observed in DNA Footprinting, antitubercular, anti-fungal and anticancer tests with these hyper π-conjugated quinoxaline derivatives. However, subtle alteration by addition of a nitro group affecting the charge distribution confers significant improvements in biological effects and binding to DNA.
Jorg Bungert - One of the best experts on this subject based on the ideXlab platform.
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combining chromatin immunoprecipitation and DNA Footprinting a novel method to analyze protein DNA interactions in vivo
Nucleic Acids Research, 2002Co-Authors: Sunghae Lee Kang, Karen F Vieira, Jorg BungertAbstract:A variety of methods are available to analyze protein– DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo Footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein–protein interactions. Furthermore, the results of in vivo Footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo Footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS Footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine β-globin promoter in MEL cells. DMS Footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the β-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein–DNA interactions in vivo.
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Combining chromatin immunoprecipitation and DNA Footprinting: a novel method to analyze protein–DNA interactions in vivo
Nucleic Acids Research, 2002Co-Authors: Sunghae Lee Kang, Karen F Vieira, Jorg BungertAbstract:A variety of methods are available to analyze protein– DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo Footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein–protein interactions. Furthermore, the results of in vivo Footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo Footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS Footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine β-globin promoter in MEL cells. DMS Footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the β-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein–DNA interactions in vivo.
Abedawn I. Khalaf - One of the best experts on this subject based on the ideXlab platform.
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Design, synthesis and antibacterial activity of minor groove binders: The role of non-cationic tail groups
European Journal of Medicinal Chemistry, 2012Co-Authors: Abedawn I. Khalaf, Claire Bourdin, David Breen, Donna Macmillan, Carol Clements, Keith Fox, Gavin Donoghue, Fraser J. Scott, Colin J. Suckling, Doreen A.t. SekiboAbstract:The design and synthesis of a new class of minor groove binder (MGBs) in which, the cationic tail group has been replaced by a neutral, polar variant including cyanoguanidine, nitroalkene, and trifluoroacetamide groups. Antibacterial activity (against Gram positive bacteria) was found for both the nitroalkene and trifluoroacetamide groups. For the case of the nitroalkene tail group, strong binding of a minor groove binder containing this tail group was demonstrated by both DNA Footprinting and melting temperature measurements, showing a correlation between DNA binding and antibacterial activity. The compounds have also been evaluated for binding to the hERG ion channel to determine whether non-cationic but polar substituents might have an advantage compared with conventional cationic tail groups in avoiding hERG binding. In this series of compounds, it was found that whilst non-cationic compounds generally had lower affinity to the hERG ion channel, all of the compounds studied bound weakly to the hERG ion channel, probably associated with the hydrophobic head groups. © 2012 Elsevier Masson SAS. All rights reserved.
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Ranking ligand affinity for the DNA minor groove by experiment and simulation.
ACS Medicinal Chemistry Letters, 2010Co-Authors: Kitiyaporn Wittayanarakul, Colin J. Suckling, Abedawn I. Khalaf, Nahoum G. Anthony, Witcha Treesuwan, Supa Hannongbua, Hasan Alniss, John Parkinson, Simon P. MackayAbstract:The structural and thermodynamic basis for the strength and selectivity of the interactions of minor groove binders (MGBs) with DNA is not fully understood. In 2003, we reported the first example of a thiazole-containing MGB that bound in a phase-shifted pattern that spanned six base pairs rather than the usual four (for tricyclic distamycin-like compounds). Since then, using DNA Footprinting, NMR spectroscopy, isothermal titration calorimetry, and molecular dynamics, we have established that the flanking bases around the central four being read by the ligand have subtle effects on recognition. We have investigated the effect of these flanking sequences on binding and the reasons for the differences and established a computational method to rank ligand affinity against varying DNA sequences.
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DNA sequence recognition by an imidazole-containing isopropyl-substituted thiazole polyamide (thiazotropsin B).
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: Andrew J. Hampshire, Colin J. Suckling, Abedawn I. Khalaf, Hannah Khairallah, Abdolrasoul H. Ebrahimabadi, Roger D. Waigh, Tom BrownAbstract:We have used DNA Footprinting and fluorescence melting experiments to study the sequence specific binding of an imidazole-containing isopropyl-substituted thiazole polyamide (thiazotropsin B) to DNA. While the parent compound (thiazotropsin A) binds to the hexanucleotide sequence ACTAGT, changing one of the N-methylpyrrole groups to N-methylimidazole changes the preferred binding sequence to (A/T)CGCG(T/A). Experiments with DNA fragments that contain variants of this sequence suggest that the ligand can also bind, with lower affinity, to sequences which differ from this by 1 bp in any position.
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DNA sequence recognition by an isopropyl substituted thiazole polyamide
Nucleic Acids Research, 2004Co-Authors: Peter L. James, Colin J. Suckling, Abedawn I. Khalaf, Roger D. Waigh, Elena E. Merkina, Tom BrownAbstract:We have used DNA Footprinting and fluorescence melting experiments to study the sequence-specific binding of a novel minor groove binding ligand (thiazotropsin A), containing an isopropyl substituted thiazole polyamide, to DNA. In one fragment, which contains every tetranucleotide sequence, sub-micromolar concentrations of the ligand generate a single footprint at the sequence ACTAGT. This sequence preference is confirmed in melting experiments with fluorescently labelled oligonucleotides. Experiments with DNA fragments that contain variants of this sequence suggest that the ligand also binds, with slightly lower affinity, to sequences of the type XCYRGZ, where X is any base except C, and Z is any base except G.
Colin J. Suckling - One of the best experts on this subject based on the ideXlab platform.
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Design, synthesis and antibacterial activity of minor groove binders: The role of non-cationic tail groups
European Journal of Medicinal Chemistry, 2012Co-Authors: Abedawn I. Khalaf, Claire Bourdin, David Breen, Donna Macmillan, Carol Clements, Keith Fox, Gavin Donoghue, Fraser J. Scott, Colin J. Suckling, Doreen A.t. SekiboAbstract:The design and synthesis of a new class of minor groove binder (MGBs) in which, the cationic tail group has been replaced by a neutral, polar variant including cyanoguanidine, nitroalkene, and trifluoroacetamide groups. Antibacterial activity (against Gram positive bacteria) was found for both the nitroalkene and trifluoroacetamide groups. For the case of the nitroalkene tail group, strong binding of a minor groove binder containing this tail group was demonstrated by both DNA Footprinting and melting temperature measurements, showing a correlation between DNA binding and antibacterial activity. The compounds have also been evaluated for binding to the hERG ion channel to determine whether non-cationic but polar substituents might have an advantage compared with conventional cationic tail groups in avoiding hERG binding. In this series of compounds, it was found that whilst non-cationic compounds generally had lower affinity to the hERG ion channel, all of the compounds studied bound weakly to the hERG ion channel, probably associated with the hydrophobic head groups. © 2012 Elsevier Masson SAS. All rights reserved.
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Ranking ligand affinity for the DNA minor groove by experiment and simulation.
ACS Medicinal Chemistry Letters, 2010Co-Authors: Kitiyaporn Wittayanarakul, Colin J. Suckling, Abedawn I. Khalaf, Nahoum G. Anthony, Witcha Treesuwan, Supa Hannongbua, Hasan Alniss, John Parkinson, Simon P. MackayAbstract:The structural and thermodynamic basis for the strength and selectivity of the interactions of minor groove binders (MGBs) with DNA is not fully understood. In 2003, we reported the first example of a thiazole-containing MGB that bound in a phase-shifted pattern that spanned six base pairs rather than the usual four (for tricyclic distamycin-like compounds). Since then, using DNA Footprinting, NMR spectroscopy, isothermal titration calorimetry, and molecular dynamics, we have established that the flanking bases around the central four being read by the ligand have subtle effects on recognition. We have investigated the effect of these flanking sequences on binding and the reasons for the differences and established a computational method to rank ligand affinity against varying DNA sequences.
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DNA sequence recognition by an imidazole-containing isopropyl-substituted thiazole polyamide (thiazotropsin B).
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: Andrew J. Hampshire, Colin J. Suckling, Abedawn I. Khalaf, Hannah Khairallah, Abdolrasoul H. Ebrahimabadi, Roger D. Waigh, Tom BrownAbstract:We have used DNA Footprinting and fluorescence melting experiments to study the sequence specific binding of an imidazole-containing isopropyl-substituted thiazole polyamide (thiazotropsin B) to DNA. While the parent compound (thiazotropsin A) binds to the hexanucleotide sequence ACTAGT, changing one of the N-methylpyrrole groups to N-methylimidazole changes the preferred binding sequence to (A/T)CGCG(T/A). Experiments with DNA fragments that contain variants of this sequence suggest that the ligand can also bind, with lower affinity, to sequences which differ from this by 1 bp in any position.
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DNA sequence recognition by an isopropyl substituted thiazole polyamide
Nucleic Acids Research, 2004Co-Authors: Peter L. James, Colin J. Suckling, Abedawn I. Khalaf, Roger D. Waigh, Elena E. Merkina, Tom BrownAbstract:We have used DNA Footprinting and fluorescence melting experiments to study the sequence-specific binding of a novel minor groove binding ligand (thiazotropsin A), containing an isopropyl substituted thiazole polyamide, to DNA. In one fragment, which contains every tetranucleotide sequence, sub-micromolar concentrations of the ligand generate a single footprint at the sequence ACTAGT. This sequence preference is confirmed in melting experiments with fluorescently labelled oligonucleotides. Experiments with DNA fragments that contain variants of this sequence suggest that the ligand also binds, with slightly lower affinity, to sequences of the type XCYRGZ, where X is any base except C, and Z is any base except G.
Sunghae Lee Kang - One of the best experts on this subject based on the ideXlab platform.
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combining chromatin immunoprecipitation and DNA Footprinting a novel method to analyze protein DNA interactions in vivo
Nucleic Acids Research, 2002Co-Authors: Sunghae Lee Kang, Karen F Vieira, Jorg BungertAbstract:A variety of methods are available to analyze protein– DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo Footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein–protein interactions. Furthermore, the results of in vivo Footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo Footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS Footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine β-globin promoter in MEL cells. DMS Footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the β-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein–DNA interactions in vivo.
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Combining chromatin immunoprecipitation and DNA Footprinting: a novel method to analyze protein–DNA interactions in vivo
Nucleic Acids Research, 2002Co-Authors: Sunghae Lee Kang, Karen F Vieira, Jorg BungertAbstract:A variety of methods are available to analyze protein– DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo Footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein–protein interactions. Furthermore, the results of in vivo Footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo Footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS Footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine β-globin promoter in MEL cells. DMS Footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the β-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein–DNA interactions in vivo.