|
|
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...
|
|
|
Function of Magnesium Ions in the Formation of Biologically-Active Nucleic Acids Structures
Espinoza Herrera, Shirly Josefina ; Štěpánek, Josef (advisor) ; Mašek, Vlastimil (referee) ; Setnička, Vladimír (referee)
ESPINOZA HERRERA S.J. - DOCTORAL THESIS - FUNCTION OF MAGNESIUM IONS IN THE FORMATION OF BIOLOGICALLY-ACTIVE NUCLEIC ACIDS STRUCTURES ____________________________________________________________________ ABSTRACT Interaction of magnesium ions, the divalent ions with the highest concentration inside living cells, with the key biomolecules of nucleic acids was studied with aim to find out structural details of the interaction mechanism and effect of the magnesium ions presence on the nucleic-acid structure and structural transitions. Raman spectroscopy was chosen as the main experimental method, some complementary measurements of UV absorption spectra were also performed. Basic analyses of the spectral series obtained as a function of temperature and/or for various concentrations of Mg2+ ions were done by means of factor analysis. Where possible the factor analysis outputs were used for fits of appropriate thermodynamic equations. For the studies, various nucleic-acid molecular models were employed, in particular complementary RNA homopolynucleotides polyA and polyU, DNA homopolynucleotides polydA and polydT and RNA 14-mer oligonucleotide representing the apical hairpin of the TAR segment of HIV-1 genomic RNA. We have confirmed via Raman spectroscopy that magnesium ions added to a solution of polyA-polyU...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
Geometry and stability study of nucleic acids distinctive single-strand motifs
Říhová, Martina ; Štěpánek, Josef (advisor) ; Mašek, Vlastimil (referee)
Subject of this thesis is the regulatory DNA segment, the so-called Serum Response Element (SRE), which possesses highly specific structural characteristics and its structural flexibility plays probably a key role in its physiological activity. Structural properties of 16-mer SRE segments capable to form hairpin structures were studied by means of accurate measurements of temperature dependent Raman spectra. Due to the close connection of oligonucleotide chain structural stability with the stacking interaction, temperature dependent Raman and UV absorption spectra were measured on dinucleotides serving as a simple model system for the stacking investigation. The measurements results processed by factor analysis provided characteristic spectral patterns and confirmed that stacking interaction depends on the base order. By fitting the thermodynamic equilibrium equations to the factor analysis outputs, the thermodynamic characteristics of the thermally induces structural changes in hairpinforming complexes were estimated and difference spectra characterizing opened and closed hairpins, which enable the further specification of the geometry of these complexes, were obtained.
|
|
|
Function of Magnesium Ions in the Formation of Biologically-Active Nucleic Acids Structures
Espinoza Herrera, Shirly Josefina ; Štěpánek, Josef (advisor) ; Mašek, Vlastimil (referee) ; Setnička, Vladimír (referee)
ESPINOZA HERRERA S.J. - DOCTORAL THESIS - FUNCTION OF MAGNESIUM IONS IN THE FORMATION OF BIOLOGICALLY-ACTIVE NUCLEIC ACIDS STRUCTURES ____________________________________________________________________ ABSTRACT Interaction of magnesium ions, the divalent ions with the highest concentration inside living cells, with the key biomolecules of nucleic acids was studied with aim to find out structural details of the interaction mechanism and effect of the magnesium ions presence on the nucleic-acid structure and structural transitions. Raman spectroscopy was chosen as the main experimental method, some complementary measurements of UV absorption spectra were also performed. Basic analyses of the spectral series obtained as a function of temperature and/or for various concentrations of Mg2+ ions were done by means of factor analysis. Where possible the factor analysis outputs were used for fits of appropriate thermodynamic equations. For the studies, various nucleic-acid molecular models were employed, in particular complementary RNA homopolynucleotides polyA and polyU, DNA homopolynucleotides polydA and polydT and RNA 14-mer oligonucleotide representing the apical hairpin of the TAR segment of HIV-1 genomic RNA. We have confirmed via Raman spectroscopy that magnesium ions added to a solution of polyA-polyU...
|
guest ::
RSS feed.