11 |
Biophysical investigation of M-DNAWood, David Owen 31 May 2005
M-DNA is a complex formed between normal double-stranded DNA and the transition metal ions Zn2+, Ni2+, and Co2+ that is favoured by an alkaline pH. Previous studies have suggested that M-DNA formation involves replacement of the imino protons of G and T bases by the transition metal ions involved in forming the complex. Owing to the conductive properties of this unique DNA conformation, it has potential applications in nanotechnology and biosensing. This work was aimed at improving existing methods and developing new methods of characterizing M-DNA. The effects of base substitutions, particularly those of G and T, were evaluated in light of the proposed structure. Differences between M-DNA conformations induced by Zn2+ and Ni2+ were also investigated with a variety of techniques and compared to the effects of Cd2+ and Mg2+ on double-stranded DNA.
M-DNA formation and stability were studied with an ethidium bromide (EtBr) based assay, M-DNA induced fluorescence quenching of DNA labelled with fluorescein and a compatible quenching molecule, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Production of monoclonal antibodies against the conformation was also attempted but was unsuccessful. The EtBr-based assay showed Ni(II) M-DNA to be much more stable than Zn(II) M-DNA as a function of pH and in the presence of ethylenediaminetetraacetic acid. Sequence-dependency and the effect of base substitutions were measured as a function of pH. With regards to sequence, d(G)nd(C)n tracts were found to form the conformation most easily. Base substitutions with G and T analogues that lowered the pKa of these bases were found to stabilize M-DNA more strongly than other base substitutions. A combination of temperature-dependant EtBr and ITC assays showed M-DNA formation to be endothermic, and therefore entropy driven. The SPR studies demonstrated many qualitative differences between Zn(II) and Ni(II) M-DNA formation, allowed characterization of Zn2+, Ni2+, Cd2+, and Mg2+ complexes with single-stranded DNA, and provided unambiguous evidence that M-DNA formation results in very little denaturation of double-stranded DNA. Specifically, the SPR study showed Ni(II) M-DNA to be more stable than Zn(II) M-DNA in the absence of transition metal ions, but also showed that Ni(II) M-DNA required higher concentrations of Ni2+ than Zn2+ to fully form the respective M-DNA conformations. Finally, quenching studies demonstrated Zn(II) M-DNA formation over a pH range from 6.5 to 8.5 provided that a Zn2+:H+ ratio of roughly 105 was maintained. The Keq for this interaction was 1.3 x 10-8 with 1.4 H+ being liberated per base bair of M-DNA formed.
These results support the proposed structural model of M-DNA, as lowering the pKa of the bases having titratable protons over the pH range studied facilitated M-DNA formation. The fact that Zn(II) M-DNA formation was observed by fluorescence quenching at any pH provided that a constant ratio of Zn2+:H+ was maintained was consistent with a simple mass-action interaction for M-DNA formation. The differences between Zn(II) and Ni(II) M-DNA formation show that although it requires a higher pH or transition metal ion concentration, Ni(II) M-DNA is more stable than Zn(II) M-DNA once formed. This difference could play an important role in applications of M-DNA which required modulation in the stability of the M-DNA conformation.
|
12 |
Biophysical investigation of M-DNAWood, David Owen 31 May 2005 (has links)
M-DNA is a complex formed between normal double-stranded DNA and the transition metal ions Zn2+, Ni2+, and Co2+ that is favoured by an alkaline pH. Previous studies have suggested that M-DNA formation involves replacement of the imino protons of G and T bases by the transition metal ions involved in forming the complex. Owing to the conductive properties of this unique DNA conformation, it has potential applications in nanotechnology and biosensing. This work was aimed at improving existing methods and developing new methods of characterizing M-DNA. The effects of base substitutions, particularly those of G and T, were evaluated in light of the proposed structure. Differences between M-DNA conformations induced by Zn2+ and Ni2+ were also investigated with a variety of techniques and compared to the effects of Cd2+ and Mg2+ on double-stranded DNA.
M-DNA formation and stability were studied with an ethidium bromide (EtBr) based assay, M-DNA induced fluorescence quenching of DNA labelled with fluorescein and a compatible quenching molecule, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Production of monoclonal antibodies against the conformation was also attempted but was unsuccessful. The EtBr-based assay showed Ni(II) M-DNA to be much more stable than Zn(II) M-DNA as a function of pH and in the presence of ethylenediaminetetraacetic acid. Sequence-dependency and the effect of base substitutions were measured as a function of pH. With regards to sequence, d(G)nd(C)n tracts were found to form the conformation most easily. Base substitutions with G and T analogues that lowered the pKa of these bases were found to stabilize M-DNA more strongly than other base substitutions. A combination of temperature-dependant EtBr and ITC assays showed M-DNA formation to be endothermic, and therefore entropy driven. The SPR studies demonstrated many qualitative differences between Zn(II) and Ni(II) M-DNA formation, allowed characterization of Zn2+, Ni2+, Cd2+, and Mg2+ complexes with single-stranded DNA, and provided unambiguous evidence that M-DNA formation results in very little denaturation of double-stranded DNA. Specifically, the SPR study showed Ni(II) M-DNA to be more stable than Zn(II) M-DNA in the absence of transition metal ions, but also showed that Ni(II) M-DNA required higher concentrations of Ni2+ than Zn2+ to fully form the respective M-DNA conformations. Finally, quenching studies demonstrated Zn(II) M-DNA formation over a pH range from 6.5 to 8.5 provided that a Zn2+:H+ ratio of roughly 105 was maintained. The Keq for this interaction was 1.3 x 10-8 with 1.4 H+ being liberated per base bair of M-DNA formed.
These results support the proposed structural model of M-DNA, as lowering the pKa of the bases having titratable protons over the pH range studied facilitated M-DNA formation. The fact that Zn(II) M-DNA formation was observed by fluorescence quenching at any pH provided that a constant ratio of Zn2+:H+ was maintained was consistent with a simple mass-action interaction for M-DNA formation. The differences between Zn(II) and Ni(II) M-DNA formation show that although it requires a higher pH or transition metal ion concentration, Ni(II) M-DNA is more stable than Zn(II) M-DNA once formed. This difference could play an important role in applications of M-DNA which required modulation in the stability of the M-DNA conformation.
|
13 |
Interactions of CPI drugs with the SV40 DNA replication origin and characterization of the porphyrin-quadruplex complexes /Han, Xiaoguang, January 1998 (has links)
Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 203-212). Available also in a digital version from Dissertation Abstracts.
|
14 |
Electrospray ionization tandem mass spectrometry methods for the analysis of DNA and DNA/drug complexesSmith, Suncerae I. 14 December 2010 (has links)
Many anticancer therapies are based on the interaction of small molecule drugs with nucleic acids, particularly DNA. Electrospray ionization tandem mass spectrometry has established itself as an irreplaceable tool for the characterization of DNA adducts produced by alkylating agents, carcinogens, and antitumor drugs, in addition to the characterization of nucleic acid post-transcriptional modifications.
ESI-MS was used to assess the non-covalent binding of a novel series of intercalating anthrapyrazoles to duplexes containing different sequences. Relative binding affinities paralleled the shift in melting point of the DNA duplexes measured from a previous study. Upon collisionally induced dissociation of the duplex/anthrapyrazole complexes, different binding strengths were discerned based on the fragmentation patterns. In addition, the interactions of a new series of sulfur-containing acridine ligands, some that functioned as alklyating mustards, with duplex DNA were also evaluated. Non-covalent and covalent binding of each ligand was determined, and the site of adduction (G > A) was revealed for the covalent modifications. The distribution of cross-linked products and mono-adducts by
psoralen analogs was also monitored by both LC-UV and IRMPD-MS methods. Reactions at 5’-TA sites were favored over 5’-AT sites. The sites of interstrand cross-linking were determined by fragmentation of the duplex/psoralen complexes by infrared multiphoton dissociation (IRMPD).
Ultraviolet photodissociation (UVPD) at 193 nm caused efficient charge reduction of deprotonated oligodeoxynucleotides via electron detachment. Subsequent CID of the charge-reduced oligodeoxynucleotides formed upon electron detachment, in a net process called electron photodetachment dissociation (EPD), resulted in a diverse array of abundant sequence ions which allowed the modification site(s) of three modified oligodeoxynucleotides to be pinpointed to a more specific location than by conventional CID.
Electron transfer dissociation (ETD) caused efficient charge reduction of multi-protonated oligonucleotides. Subsequent CAD of the charge-reduced oligonucleotides formed upon electron transfer, in a net process termed electron transfer collision activated dissociation (ETcaD), resulted in rich backbone fragmentation, with a marked decrease in the abundance of base loss ions and internal fragments. ETcaD of an oligonucleotide duplex resulted in specific backbone cleavages, with conservation of weaker non-covalent bonds. In addition, IRMPD and UVPD were used to activate charge-reduced oligonucleotides formed upon electron transfer. ET-IRMPD afforded tunable characterization of the modified DNA and RNA, allowing for modified bases to be directly analyzed. ET-UVPD promoted higher energy backbone fragmentation pathways and created the most diverse MS/MS spectra. The numerous products generated by the hybrid MS/MS techniques (ETcaD, ET-IRMPD, and ET-UVPD) resulted in specific and extensive backbone cleavages which allowed for the modification sites of multiple oligonucleotides to be pinpointed. / text
|
15 |
Crystallographic studies on DNA in complex with antibiotics and effects of anisotropic scaling on structure solution / Kristallstrukturanalyse von DNA in Komplexen mit Antibiotika und Effekte anisotroper Skalierung auf die StrukturlösungPfoh, Roland 30 October 2008 (has links)
No description available.
|
Page generated in 0.0534 seconds