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Vibrational modes of nucleic acids components characterized by means of variable proton-deuterium exchange
Zoul, David ; Štěpánek, Josef (advisor) ; Šloufová, Ivana (referee)
Although the nucleon acid oscillatory spectra have been studied for a long period of time, many of oscillating modes have not been properly explained regarding their geometric sensitivity and weak binding. One of the possibilities how to acquire experimental data for interpretation of theese modes is to confront oscillating spectra of isotopes. The simplest is proton - deutron exchange. When molecules dissolve in heavy water they rapidly exchange the heteroatoms, but this is simultaneous for more stages, which leads to dynamic equilibrium state among various isotope forms. Statistic analysis of temporal progression of Raman spectra give us the possibility to analyze the spectra and to obtain unique experimental data. This technique is very promising for more complex nucleon acid segments to gain more structural information. This diploma thesis represents pilot study of introduced methodics. The objective is to implement adaptation of Raman spectrometr for given types of expiments and undertake first series of measurements. The results should clarify application bounds of proposed method refering to sensitivity, time constant of incident isotope exchange, severity of read-out in comparasion of achieved accuracy of the measurement.
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Structural flexibility of regulatory DNA element
Řezáčová, Barbora ; Štěpánek, Josef (advisor) ; Rosenberg, Ivan (referee)
Transcription of the genetic code is controlled by numerous proteins. Some of them, so-called transcription factors, bind to certain DNA regions possessing anomalous properties due to their specific and highly conservative base sequence. One of these DNA segments is SRE (Serum Response Element). The diploma work is linked to results of the previous study indicating that the structural flexibility of this segment, related to the CArG box sequence, plays a key role in its physiological activity. Our work is devoted to study of this DNA segment and its model sequential variants. The approach of accurate measurements of temperature dependent UV absorption spectra and their consecutive analysis by means of factor analysis techniques and difference spectra construction, the main part of which was also developed within the framework of this work, was employed. The results indicate that separate chains of SRE with various length form surprisingly stable hairpins, which in some cases prevail even in mixtures of both chains of the SRE segment over duplexes. By using a fitting to thermodynamic equilibrium equations, thermodynamic characteristics of temperature induced association and dissociation transitions of complexes, both intermolecular (duplexes) and intramolecular (hairpins), were obtained.
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Microcalorimetric determination of RNA triplex thermodynamic stability
Říhová, Martina ; Štěpánek, Josef (advisor) ; Kočišová, Eva (referee)
Nazcv prace: Mikrokalorimelricke slanovcni termodynamicke stability triplexu RNA Autor: Martina Rihova Kaledra (ustav): Fyzikalni ustav UK Vcdouci bakalafske prace: Prof. RNDr. Josef Stcpanck, Csc. e-mail vedouciho: stepjosiyj;karlov.mff.cuni.cz Abstrakt: Prace je zamC'fena na urcovani termodynamickych charaktcristik duplexu a triplexu ribonukleove kyscliny. Rozpad a v/nik komplexu hyl sledovan na diferencnim skcnovacim mikrokalorimelru. Jako vzorky byly pouzity smesne ro/toky syntelicky pfipravenych homopolymerii RNA, polyA a polyU, o ruznych stcchiometrickych pomerech. Z namcfenych dal byly urceny termodynamickc paramclry komplexu, tcdy odpovidajici zmeny entalpie AH a entropic AS a teplola tani komplexu. Vysledky ukazaly, /e pfi techto mcfcnich dochazi k systematickc /mene namerenych charakteristik pfi opakovanych scanech. Tento jcv patrnc souvisi s intcrakci RNA s povrchem mefici kyvcty a jc tedy zavazny z hlediska aplikovatclnosli scanovaci mikrokalorimctric pro pfesna kvantitativni tennodynamicka studia ribonukleove kyscliny. Zmcny teploty tani zpusobenc pridanim hofciku do rozloku uka/aly, zc jeho ucinkem so pfi pfipojeni ifctiho vlakna k duplexu RNA komplex stabilizuje. Klicova slova: triplex RNA, duplex RNA, mikrokalorimetric, termodynamickc charakteristiky, teplota tani. Title: Microcalorimetric...
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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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