The Experts below are selected from a list of 435 Experts worldwide ranked by ideXlab platform
William H. Gmeiner - One of the best experts on this subject based on the ideXlab platform.
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Cytarabine-induced destabilization and bending of a model Okazaki fragment.
Nucleosides Nucleotides & Nucleic Acids, 1999Co-Authors: William H. Gmeiner, Alan Skradis, Richard T. Pon, Jinqian LiuAbstract:The effects of cytarabine on the structural and thermodynamic properties of an Okazaki fragment were investigated using UV Hyperchromicity and 2D 1H NMR. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 degrees C to 42.4 degrees C at 1.33 x 10(-5) M. Cytarabine also markedly increased the bend angle of the Okazaki fragment duplex from 20 degrees to 42 degrees. Changes to the structures and stabilities of Okazaki fragments may cause the biological effects of cytarabine.
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Cytarabine-induced destabilization of a model Okazaki fragment.
Nucleic Acids Research, 1998Co-Authors: William H. Gmeiner, Alan SkradisAbstract:Cytarabine is a potent anticancer drug that interferes with elongation of the lagging strand at the replication fork during DNA synthesis. The effects of cytarabine substitution on the structural and thermodynamic properties of a model Okazaki fragment were investigated using UV Hyperchromicity and 1H NMR spectroscopy to determine how cytarabine alters the physicochemical properties of Okazaki fragments that are intermediates during DNA replication. Two model Okazaki fragments were prepared corresponding to a primary initiation site for DNA replication in the SV40 viral genome. One model Okazaki fragment consisted of five ribo- and seven deoxyribonucleotides on the hybrid strand, together with its complementary (DNA) strand. The second model Okazaki fragment was identical to the first with the exception of cytarabine substitution for deoxycytidine at the third DNA nucleotide of the hybrid strand. Thermodynamic parameters for the duplex to single strand transition for each model Okazaki fragment were calculated from the concentration dependence of the T m at 260 nm. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 to 42.4 degrees C at a concentration of 1.33 x 10(-5) M. The free energy for the duplex to single strand transition was 1.2 kcal/mol less favorable for the cytarabine-substituted Okazaki fragment relative to the control at 37 degrees C. Analysis of the temperature dependence of the imino1H resonances for the two duplexes demonstrated that cytarabine specifically destabilized the DNA:DNA duplex portion of the model Okazaki fragment. These results are consistent with inhibition of lagging strand DNA synthesis by cytarabine substitution resulting from destabilization of the DNA:DNA duplex portion of Okazaki fragments in vivo .
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Synthesis of 5'-O-(4,4'-Dimethoxytrityl)-2'-O-(tert-butyldimethylsilyl)-5-fluorouridine 3'-(Cyanoethyl N,N-diisopropylphosphoramidite) and Its Use in the Synthesis of RNA
The Journal of Organic Chemistry, 1994Co-Authors: William H. Gmeiner, Parag V. Sahasrabudhe, Richard T. PonAbstract:5-Fluorouridine (FUrd) has been successfully converted to 5'-O-(4,4'-dimethoxytrityl)-2'-O-(tertbutyldimethylsilyl)-5-fluorouridine 3'-(cyanoethyl N,N-diisopropyl(phosphoramidite) by methods similar to those employed for the preparation of other ribonucleoside phosphoramidites. The desired product, as well as its precursors and the incorrect regioisomers, are prepared and fully characterized by 1 H NMR, 19 F NMR, 31 P NMR, FAB-MS, UV absorbance, elemental analysis, and melting points. The purified material is used for the production of RNA by solid-phase methods. Coupling yields of 94% are obtained with this material. The resulting RNA is readily purified and used for biophysical studies. The self-complemetary RNA decamer 5'-rGCGAAU(FU)CGC is prepared and purified. The material adopts an A-form duplex in solution at neutral pH as characterized by CD spectroscopy. The thermal stability of the duplex is similar to the parent duplex oligoribonucleotide prepared with uridine instead of 5-fluorouridine. The control duplex has a T m of 53.8 and the FUrd substituted duplex has a T m of 56.6 ×b0° C as determined by UV Hyperchromicity at 260nm. The CD spectra and UV Hyperchromicity data for the duplex oligoribonucleotide containing FUrd have a slight pH dependence indicating that ionization of FUrd has a slight but measurable impact on RNA duplex stability. 1D NMR spectroscopy of the imino hydrogens for this duplex in H 2 O solution confirms the formation of Watson-Crick base pairs. The imino hydrogen resonance for the FUrd-A base pair is moved downfield and broadened compared with a control duplex oligoribonucleotide as a consequence of the electron-withdrawing inductive effect of fluorine
Alan Skradis - One of the best experts on this subject based on the ideXlab platform.
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Cytarabine-induced destabilization and bending of a model Okazaki fragment.
Nucleosides Nucleotides & Nucleic Acids, 1999Co-Authors: William H. Gmeiner, Alan Skradis, Richard T. Pon, Jinqian LiuAbstract:The effects of cytarabine on the structural and thermodynamic properties of an Okazaki fragment were investigated using UV Hyperchromicity and 2D 1H NMR. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 degrees C to 42.4 degrees C at 1.33 x 10(-5) M. Cytarabine also markedly increased the bend angle of the Okazaki fragment duplex from 20 degrees to 42 degrees. Changes to the structures and stabilities of Okazaki fragments may cause the biological effects of cytarabine.
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Cytarabine-induced destabilization of a model Okazaki fragment.
Nucleic Acids Research, 1998Co-Authors: William H. Gmeiner, Alan SkradisAbstract:Cytarabine is a potent anticancer drug that interferes with elongation of the lagging strand at the replication fork during DNA synthesis. The effects of cytarabine substitution on the structural and thermodynamic properties of a model Okazaki fragment were investigated using UV Hyperchromicity and 1H NMR spectroscopy to determine how cytarabine alters the physicochemical properties of Okazaki fragments that are intermediates during DNA replication. Two model Okazaki fragments were prepared corresponding to a primary initiation site for DNA replication in the SV40 viral genome. One model Okazaki fragment consisted of five ribo- and seven deoxyribonucleotides on the hybrid strand, together with its complementary (DNA) strand. The second model Okazaki fragment was identical to the first with the exception of cytarabine substitution for deoxycytidine at the third DNA nucleotide of the hybrid strand. Thermodynamic parameters for the duplex to single strand transition for each model Okazaki fragment were calculated from the concentration dependence of the T m at 260 nm. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 to 42.4 degrees C at a concentration of 1.33 x 10(-5) M. The free energy for the duplex to single strand transition was 1.2 kcal/mol less favorable for the cytarabine-substituted Okazaki fragment relative to the control at 37 degrees C. Analysis of the temperature dependence of the imino1H resonances for the two duplexes demonstrated that cytarabine specifically destabilized the DNA:DNA duplex portion of the model Okazaki fragment. These results are consistent with inhibition of lagging strand DNA synthesis by cytarabine substitution resulting from destabilization of the DNA:DNA duplex portion of Okazaki fragments in vivo .
Sven Klussmann - One of the best experts on this subject based on the ideXlab platform.
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stereospecificity of oligonucleotide interactions revisited no evidence for heterochiral hybridization and ribozyme dnazyme activity
PLOS ONE, 2015Co-Authors: Kai Hoehlig, Lucas Bethge, Sven KlussmannAbstract:A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using Hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.
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Achiral PNA hybridizes to both d- and l-RNA/DNA.
2015Co-Authors: Kai Hoehlig, Lucas Bethge, Sven KlussmannAbstract:Hybridization of achiral PNA and anti-parallel complementary (A) d-RNA, (B) l-RNA, (C) d-DNA, and (D) l-DNA (3 μM each in 10 mM phosphate buffer, pH 7.4, 100 mM NaCl) was analyzed by measuring temperature-dependent Hyperchromicity at 260 nm. Mean of three melting ramps (25°C to 95°C) is given as normalized absorption A / Amax at 260 nm. First derivative is shown as dotted line. Data are representative of three independent experiments.
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Hybridization of anti-parallel complementary oligonucleotides is stereospecific.
2015Co-Authors: Kai Hoehlig, Lucas Bethge, Sven KlussmannAbstract:Schematic representation of (A) hammerhead ribozyme and (B) DNAzyme in complex with the target RNA sequence (green). Hydrolysis sites are indicated by arrows. To analyze (potential) hybridization of enantiomeric, anti-parallel complementary oligonucleotides (3 μM each in 10 mM phosphate buffer, pH 7.4, 100 mM NaCl) temperature-dependent Hyperchromicity at 260 nm was measured. (C) d-RNA and (D) l-RNA with anti-parallel complementary d-RNA (red) and l-RNA (blue). (E) d-RNA and (F) l-RNA with anti-parallel complementary d-DNA (red) and l-DNA (blue). (G) d-DNA and (H) l-DNA with anti-parallel complementary d-DNA (red) and l-DNA (blue). Mean of three melting ramps (25°C to 95°C) is given as normalized absorption A / Amax at 260 nm. First derivative is shown as dotted line. Data are representative of three independent experiments.
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Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity
2015Co-Authors: Kai Hoehlig, Lucas Bethge, Sven KlussmannAbstract:A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using Hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.
Wahid Ali Khan - One of the best experts on this subject based on the ideXlab platform.
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preferential recognition of catechol estrogen modified dna by circulating autoantibodies in cancer patients
Biochimie, 2013Co-Authors: Wahid Ali Khan, Safia HabibAbstract:Abstract Catecholestrogens [4-hydroxyestradiol (4-OHE 2 )] have been implicated in human carcinogenesis, although the mechanism remains unestablished. In this study pUC 18 plasmid DNA was modified with 4-OHE 2 and nitric oxide (NO). The modification induced in native DNA exhibited Hyperchromicity, single strand breaks, damage to restriction sites, modification of bases, decrease in Tm and change in ellipticity. Modified DNA was found to be highly immunogenic in experimental animal, eliciting high titer antibodies. Circulating cancer autoantibodies showed preferable recognition of 4-OHE 2 -NO-DNA over native form ( p 2 -NO-DNA antibodies as a probe. Preferential recognition of 4-OHE 2 -NO-DNA by cancer autoantibodies coupled with enhanced binding of induced antibodies to DNA isolated from cancer patients is an indicative of oxidative stress induced DNA damage in cancer. Possible involvement of unique epitopes on modified DNA in cancer autoantibody induction has been suggested.
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Catechol-estrogen modified DNA: a better antigen for cancer autoantibody.
Archives of biochemistry and biophysics, 2007Co-Authors: Wahid Ali Khan, Khursheed Alam, MoinuddinAbstract:Estrogens are known mutagenic and carcinogenic risk factors. Non-enzymatic oxidation of catechol-estrogens in the presence of copper is reported to generate reactive oxygen species (ROS) that can cause DNA damage. We show that DNA modification in the presence of 4-hydroxyestradiol (4-OHE(2)) and copper (Cu-II) results in single and double strand breaks, base modification, Hyperchromicity and change in ellipticity. Modified DNA (4-OHE(2)-Cu(II)-DNA) was highly immunogenic in experimental animals. Induced anti-4-OHE(2)-Cu(II)-DNA antibodies were effectively used as a probe for detecting oxidative lesions in human genomic DNA and for the estimation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the urine of cancer patients. Circulating antibodies from cancer patients showed high binding to 4-OHE(2)-Cu(II)-DNA as compared to native DNA. Our results imply that interaction of catechol-estrogen and copper leads to the production of potent ROS, capable of causing DNA damage, thus playing an important role in carcinogenesis. The modified DNA presents unique epitopes which may be one of the factors for autoantibody induction in cancer.
Richard T. Pon - One of the best experts on this subject based on the ideXlab platform.
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Cytarabine-induced destabilization and bending of a model Okazaki fragment.
Nucleosides Nucleotides & Nucleic Acids, 1999Co-Authors: William H. Gmeiner, Alan Skradis, Richard T. Pon, Jinqian LiuAbstract:The effects of cytarabine on the structural and thermodynamic properties of an Okazaki fragment were investigated using UV Hyperchromicity and 2D 1H NMR. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 degrees C to 42.4 degrees C at 1.33 x 10(-5) M. Cytarabine also markedly increased the bend angle of the Okazaki fragment duplex from 20 degrees to 42 degrees. Changes to the structures and stabilities of Okazaki fragments may cause the biological effects of cytarabine.
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Synthesis of 5'-O-(4,4'-Dimethoxytrityl)-2'-O-(tert-butyldimethylsilyl)-5-fluorouridine 3'-(Cyanoethyl N,N-diisopropylphosphoramidite) and Its Use in the Synthesis of RNA
The Journal of Organic Chemistry, 1994Co-Authors: William H. Gmeiner, Parag V. Sahasrabudhe, Richard T. PonAbstract:5-Fluorouridine (FUrd) has been successfully converted to 5'-O-(4,4'-dimethoxytrityl)-2'-O-(tertbutyldimethylsilyl)-5-fluorouridine 3'-(cyanoethyl N,N-diisopropyl(phosphoramidite) by methods similar to those employed for the preparation of other ribonucleoside phosphoramidites. The desired product, as well as its precursors and the incorrect regioisomers, are prepared and fully characterized by 1 H NMR, 19 F NMR, 31 P NMR, FAB-MS, UV absorbance, elemental analysis, and melting points. The purified material is used for the production of RNA by solid-phase methods. Coupling yields of 94% are obtained with this material. The resulting RNA is readily purified and used for biophysical studies. The self-complemetary RNA decamer 5'-rGCGAAU(FU)CGC is prepared and purified. The material adopts an A-form duplex in solution at neutral pH as characterized by CD spectroscopy. The thermal stability of the duplex is similar to the parent duplex oligoribonucleotide prepared with uridine instead of 5-fluorouridine. The control duplex has a T m of 53.8 and the FUrd substituted duplex has a T m of 56.6 ×b0° C as determined by UV Hyperchromicity at 260nm. The CD spectra and UV Hyperchromicity data for the duplex oligoribonucleotide containing FUrd have a slight pH dependence indicating that ionization of FUrd has a slight but measurable impact on RNA duplex stability. 1D NMR spectroscopy of the imino hydrogens for this duplex in H 2 O solution confirms the formation of Watson-Crick base pairs. The imino hydrogen resonance for the FUrd-A base pair is moved downfield and broadened compared with a control duplex oligoribonucleotide as a consequence of the electron-withdrawing inductive effect of fluorine