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Preparation of DNA with local DNA structures
Lofítková, Ellen ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
In this thesis I focused on quadruplexes and cruciforms formed by DNA. Plasmids pBluescript and others derived from it by inserting oligonucleotid sequences were studied. Sequences forming quadruplexes and cruciforms were found by in silico analysis by QGRS Mapper and Palindrome analyser. Plasmids were transformed into E. coli, isolated and then cleaved with enzymes S1 nuclease and restriction endonuclease ScaI. Cleaving with S1 nuclease predicated presence of local structure. Combined S1/ScaI cleavage did not bring satisfying results due to lost of DNA during purification.
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Analysis of local structures in DNA molecules
Nyczová, Adéla ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
Local DNA structures are alternative DNA conformations which can be formed aside from typical B-DNA conformation. These structures often play pivotal roles in regulation of basic biological processes, such as DNA replication, transcription or binding of specific ligands. This biological significance makes alternative DNA secondary structures a potential drug target. In this diploma thesis, local structures in genomes of viruses from Flaviviridae and Retroviridae families are analysed using bioinformatics tools. Furthermore, these structures are visualised using atomic force microscopy.
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Quadruplex Detection in DNA Sequences
Sečka, Martin ; Lexa, Matej (referee) ; Martínek, Tomáš (advisor)
This master's thesis focuses on search for sequences potentially forming quadruplexes in DNA sequences, their visualization and filtration. For this purpose a web application was created in cooperation with the Institute of biophysics of Academy of science of Czech republic. This application uses a newly created algorithm able to find all possible formations of quadruplex within given input sequence. Design and implementation of this algorithm are also part of this thesis.
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Non-resonant Raman Spectroscopic Study of Guanine Quadruplex Structures
Golan, Martin ; Mojzeš, Peter (advisor) ; Mašek, Vlastimil (referee)
Parts of human telomere sequences containing at least 4 guanine subsequences show the ability to form intrastrand quadruplexes of remarkable conformational diversity. Former studies using conventional Raman spectroscopy have revealed that the sequence G3(TTAG3)3 at milimolar concentrations in phosphate buffer solution doped with Na+ ions (ionic strength 150 mM) adopts antiparallel conformation regardless of the length of standing at room temperature or annealing, whereas K+ ions cause gradual transition to "3+1" or even parallel conformation. On the other hand, measurements carried out upon sequence AG3(TTAG3)3 at similar concentrations using Photonic Crystal Fibre-enhanced Raman Spectroscopy (PCFRS) suggest that in the respective presence of both Na+ and K+ (ionic strength 100 mM), a parallel structure is adopted. The hereby presented work employs conventional Raman spectroscopy and Drop Coating Deposition Raman spectroscopy to examine the sequence AG3(TTAG3)3 at concentrations ranging from units to hundreds of milimoles in strands. It concludes that the structure adopted in the presence of Na+, resp. K+ ions is antiparallel, resp. "3+1", and doesn't change over time despite both long standing and annealing. Two hypotheses about the cause of the differences between the results obtained by PCFRS and...
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Conformational transitions and stability of quadruplex nucleic acids
Johanovská, Zuzana ; Mojzeš, Peter (advisor) ; Profant, Václav (referee)
The work deals with guanine quadruplexes, i.e., non-canonical four-stranded structures of nucleic acids rich in repetitive guanine sequences with potential biological and nanotechnological significance. The first part summarizes basic information about the structure and topology of quadruplexes, in the second part, the present knowledge about the quadruplexes in vivo are overviewed. The third part is devoted to experimental methods used in the present study, namely Raman spectroscopy, circular dichroism, and absorption spectroscopy. The next sections are devoted to the acquisition and treatment of your own experimental results. Five sequentially related oligonucleotides differing in the bases in a single-member loop were investigated. The effect of concentration of oligonucleotide and potassium ions K+ on the stability of these quadruplexes was investigated by Raman spectroscopy, and an unusually high thermal stability was observed. Other experiments included measurements of CDs and absorption spectra at lower oligonucleotide concentrations, two different potassium ion concentrations, and a comparison of the stability of individual modifications with the previously published results. Furthermore, the possible interaction with the cationic porphyrin CuTMPyP4, similar to the interaction with the antiparallel...
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Biologically important non-canonical structures of nucleic acids in complexes with cationic porphyrins
Palacký, Jan ; Mojzeš, Peter (advisor) ; Setnička, Vladimír (referee) ; Víglaský, Viktor (referee)
Guanine quadruplexes are a class of unusual nucleic acids conformations based on stacked planar guanine tetrads stabilized via Hoogsteen pairing and cation coordination. They are implicated in numerous cellular processes including replication, recombination or transcription. Guanine quadruplexes are widespread within the human genome but their occurrence is highest in the single stranded guanine-rich regions at telomeres. Telomeric guanine quadruplexes are gaining growing interest due to their ability to inhibit the activity of the telomerase enzyme, which is responsible for the proliferation of tumor cells. Specifically, we investigated the conformational polymorphism of the human core telomeric sequence G3(TTAG3)3 conditioned by the concentration of DNA, metal cations (K+ , Na+ ) and/or annealing. Raman spectroscopy was employed as the primary method for this study because, unlike common spectroscopic methods, it allowed us to monitor the quadruplex structure at very high DNA concentrations mimicking molecular crowding conditions in the cell. We demonstrate that the G3(TTAG3)3 quadruplex switch between the antiparallel and parallel strand alignment as a function of nucleoside and potassium concentration. In addition, we demonstrate that cationic porphyrins can be used as sensitive probes of the quadruplex...
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Analysis of local structures in DNA molecules
Nyczová, Adéla ; Smetana, Jan (referee) ; Brázda, Václav (advisor)
Local DNA structures are alternative DNA conformations which can be formed aside from typical B-DNA conformation. These structures often play pivotal roles in regulation of basic biological processes, such as DNA replication, transcription or binding of specific ligands. This biological significance makes alternative DNA secondary structures a potential drug target. In this diploma thesis, local structures in genomes of viruses from Flaviviridae and Retroviridae families are analysed using bioinformatics tools. Furthermore, these structures are visualised using atomic force microscopy.
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Concentration-dependent topology of telomere DNA quadruplexes
Bokach, Anton ; Římal, Václav (advisor) ; Štěpánek, Josef (referee)
Telomeres contain repetitive DNA sequences and play a key role in cellular ageing and cancer. Human telomeric DNA contains guanine-rich sequences, such as (GGGTTA)n, that are capable of four-stranded quadruplex structures assembling. Quadruplexes can adopt various topologies depending on the ionic environment, concentration, thermal treatment and other factors. Those topologies include parallel, anti-parallel and hybrid (3+1) types. We explored the effect of dilution and annealing of two human telomere sequences (21 and 22 nucleotide long) in a potassium-containing solution by nuclear magnetic reso- nance (NMR) and Raman spectroscopies. We found that multimolecular complexes with parallel orientation of the DNA strands, which are formed at high DNA concentration (≈ 10 mM), persist even after strong dilution. They are disrupted only by thermal an- nealing. Furthermore, annealing led to the creation of a unimolecular G-quadruplex that appeared to be in (3 + 1) conformation. The (3 + 1) conformation of G-quadruplex is the most populated monomeric quadruplex structure present in the solution, regardless of oligonucleotide concentration. We examined the differences between quadruplex structures in crowded and diluted solutions, as well as, before and after annealing. The presented thesis brings new in- sights...
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