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Study of nucleic-acid structure and interactions by resonance Raman scattering
Klener, Jakub ; Štěpánek, Josef (advisor)
Despite the decades of intensive research, nucleic acids represent still a permanent object of structural studies. Within the framework of the doctoral work, the apparatus for measurement of UV excited resonance Raman spectra (UV RRS) was built up and optimized. A realistic and complex interpretation table was prepared based on analysis of published data and extensive series of UV RRS measurements on NA model structures, mononucleotides, and polynucleotides. The established methodology was verified when applied in several structural studies of nucleic acids, mainly the study of the influence of magnesium ions on the equilibrium between duplexes and triplexes formed by PolyA and PolyU homopolynucleotides, a study of temperature-induced structural changes in DNA double helix and DNA hairpin, and investigation of slow structural transitions of guanine quadruplexes induced by the presence of potassium ions. The results of the test measurements and the above-mentioned studies have shown that the created methodology for studying UV RRS of nucleic acids brings most of the expected benefits of the resonance excitation: the possibility of Raman scattering measurements at the same concentrations as in the case of UV absorption, high sensitivity to fine temperature-induced structural changes and good interpretability...
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Study of nucleic-acid structure and interactions by resonance Raman scattering
Klener, Jakub ; Štěpánek, Josef (advisor) ; Michl, Martin (referee) ; Vlčková, Blanka (referee)
Despite the decades of intensive research, nucleic acids represent still a permanent object of structural studies. Within the framework of the doctoral work, the apparatus for measurement of UV excited resonance Raman spectra (UV RRS) was built up and optimized. A realistic and complex interpretation table was prepared based on analysis of published data and extensive series of UV RRS measurements on NA model structures, mononucleotides, and polynucleotides. The established methodology was verified when applied in several structural studies of nucleic acids, mainly the study of the influence of magnesium ions on the equilibrium between duplexes and triplexes formed by PolyA and PolyU homopolynucleotides, a study of temperature-induced structural changes in DNA double helix and DNA hairpin, and investigation of slow structural transitions of guanine quadruplexes induced by the presence of potassium ions. The results of the test measurements and the above-mentioned studies have shown that the created methodology for studying UV RRS of nucleic acids brings most of the expected benefits of the resonance excitation: the possibility of Raman scattering measurements at the same concentrations as in the case of UV absorption, high sensitivity to fine temperature-induced structural changes and good interpretability...
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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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Thermodynamics of complex formation by miRNA
Vacková, Anežka ; Štěpánek, Josef (advisor) ; Mojzeš, Peter (referee)
The bachelor thesis concerns microRNA (miRNA), short RNA molecules that mediate post-transcriptional regulation of gene expression by binding to a target mRNA. They usually form extended duplexes consisted of short tracks of Watson-Crick basepairs separated by internal loops, mismatched pairs, or bulges. The thesis represents a study of the effect of structural anomalies, the internal loop and the bulge, on the structural stability miRNA Let7 complex with its target LCS1 sequence at mRNA lin-41 from Caenorhabditis Elegans. The task was solved by recording temperature dependent UV absorption spectra of solutions containing mixtures of the mentioned above RNA sequences and their mutated analogs, analysis of the spectral changes by using a singular decomposition method followed by fitting to a thermodynamic model. The determined thermodynamic characteristics of the complexes and spectral changes caused by a temperature increase or the complex dissociation are the outcome of the work.
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miRNA complexes with the target RNA sequence
Homolka, Radim ; Štěpánek, Josef (advisor) ; Římal, Václav (referee)
miRNAs are short non-coding RNA molecules which play role in post-transcriptional regulation of gene expression. Thus, miRNAs are related to a variety of physiological processes and diseases, e.g. some tumours or viral infections. In this diploma thesis we studied temperature-induced dissociation of complexes that simulated pairing of miRNA with target mRNA. For these purposes we used UV absorption spectroscopy and Raman spectroscopy. Measured spectra were then treated by means of a factor analysis. The aim of the thesis was to determine thermodynamic parameters of the complexes dissociation and to clarify how inner loops and bulges affect the stability of oligonucleotide duplexes. Also, we have tested the suitability of the used approach for study of oligonucleotide complexes. We have shown that factor analysis of temperature dependent UV absorption spectra is a proper base to determine thermodynamic parameters of the duplex dissociation. Raman spectra were affected by large errors, nevertheless they enabled to observe some structural changes related to the transition. It has been shown that the bulge loop present in the studied complex reduces both its flexibility and the energetic preference of its formation.
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Spectroscopic Study of miRNA Structure and Interactions
Homolka, Radim ; Štěpánek, Josef (advisor) ; Maloň, Petr (referee)
MiRNA are short RNA molecules that mediate post-transcriptional regulation of gene expression by binding to a target mRNA. In this thesis, some structural properties and thermodynamic parameters (melting temperature, enthalpy ∆H and entropy ∆S changes connected with the transition between particular conformations) of oligonucleotides simulating complex of miRNA and mRNA, were characterized by using spectroscopic methods and a gel electrophoresis. Temperature dependences of UV and Raman spectra were measured, the results treated by means of a factor analysis, and the abovementioned thermodynamic parameters determined by a fit to a feasible model. The outputs have demonstrated the capability of the used methods to determine thermodynamic and structural properties of non-canonical RNA complexes. Powered by TCPDF (www.tcpdf.org)
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In situ study of nuclear acids interactions key for gene expression and therapy based on its silencing
Špringer, Tomáš ; Štěpánek, Josef (advisor) ; Brynda, Eduard (referee) ; Vorlíčková, Michaela (referee)
In this doctoral thesis we study novel analogues based on R06 aptamers and targeting TAR hairpins of the HIV virus by means of surface plasmon resonance biosensor, which allows for sensitive and real-time monitoring of molecular interactions. We investigate seven different modifications placed at nine different positions on the R06 aptamer in order to find out their applicability in the construction of efficient and stable anti-TAR oligonucleotides. We also determine which positions are suitable for substitutions with a modification and interpret the results in the context of the local nucleotide geometries and interactions in the TAR/anti-TAR complex. In this doctoral thesis we further develop a new fluidic system. This fluidic system eliminates sample dispersion and intermixing effects and thus enables accurate monitoring of molecular interactions on the surface of an SPR chip. We also characterize experimental conditions on the surface of an oligonucleotide chip and their relations towards bio-molecular assays. Specifically, we study the shielding effect of monovalent and divalent cations, which are crucial for the interaction of negatively charged oligonucleotides.
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Molecular mechanism of DNA regulatory segment recognition by MADS box family transcription factors
Profantová, Barbora ; Štěpánek, Josef (advisor) ; Jelínek, Otakar (referee) ; Manfait, Michel (referee)
The thesis deals with physico-chemical properties of the MADS box, binding domain of transcription factors, which are important for the formation of complexes with the DNA regulatory segment bearing the CArG box. The study was performed also on model oligopeptides, selected segments of the MADS box and their analogues with a point mutation. A wide range of spectroscopic techniques was employed, namely absorption, circular dichroism, fluorescence and Raman spectroscopies. Advanced approaches including multivariate methods were used for data processing. The three tyrosines of the MADS box located in amino-acid vicinities of different charge and hydrophobicity, were used as intrinsic spectroscopic probes. The obtained characteristics of the MADS box and its segments structural arrangement, flexibility and acid-base equilibria are the main results of the work.
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Spectroscopic study of key biomolecular interactions for therapeutic applications of modified oligonucleotids
Kříž, Martin ; Štěpánek, Josef (advisor) ; Bednárová, Lucie (referee) ; Mašek, Vlastimil (referee)
Title: Spectroscopic study of key biomolecular interactions for therapeutic applications of modified oligonucleotides Author: Martin Kříž Department: Institute of Physics of Charles University Supervisor: Prof. RNDr. Josef Štěpánek, CSc., Institute of Physics of Charles University Abstract: This dissertation thesis is focused on study of biomolecular interactions that are crucial for therapeutic applications of modified oligonucleotides. Interactions of nucleic acids, in particular hybrid DNA: RNA duplexes with fully complementary sequences, as well as DNA: RNA duplexes with a central mismatch were studied. The molecular model was created. With this model the influence of five different chemical modifications in the DNA strand to its binding ability was studied. Structural changes were characterized by Raman spectroscopy using a precise differential and double differential spectra. These data were supplemented by thermodynamic parameters computed from melting curves obtained by UV absorption spectroscopy. Furthermore, the Raman spectroscopy was applied on study of conformation changes in the RNase L protein. These changes in the conformation occur after ligand binding, which in this case, either natural or modified short oligonucleotide. Measurement of RNase L was performed using a special experimental...
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