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Investigation of the Properties of Novel Contrast Agents for Magnetic Resonance Imaging
Plšek, Martin ; Římal, Václav (advisor) ; Březina, Václav (referee)
Abstract: Contrast agents for magnetic resonance imaging frequently employ organic com- plexes of paramagnetic ions. The present work studies rotation of phoshonate group in LnIII complexes of monoethylester-P of 1,4,7,10-tetraazacyclododecane-4,7,10-tris(karboxyme- thyl)-1-methylphosphonic acid (Lndo3apOEt ), specifically Lado3apOEt and Cedo3apOEt . 31 P and 1 H NMR spectra were measured in a wide temperature range. Transverse relaxation times and chemical shifts of the two 31 P spectral lines which undergo mutual chemical exchange were estimated. The phosphorus spectra were then subjected to line-shape analysis, thus cha- racterizing the chemical exchange connected to phosphonate rotation. This process yielded temperature dependence of both rate coefficients and relative populations between the two di- astereoisomers for both studied molecules. The termodynamic parameters were obtained and compared with similar complexes with phosphonate rotation. The knowledge of the detailed kinetics can later lead to improvements of the efficiency of the contrast agents. Keywords: nuclear magnetic resonance, MRI contrast agent, kinetics of conformational ex- change
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The thermodynamics of DNA-hairpin formation
Sgallová, Ráchel ; Římal, Václav (advisor) ; Profant, Václav (referee)
Serum response factor (SRF) is a transcription factor which binds to a highly conserved DNA sequence called the CArG box. According to the nucleotide sequence of CArG box it could form a hairpin structure or a cruciform. In this master thesis, the structure of the CArG box in a human gene c-fos was studied by nuclear magnetic resonance. 1 H spectra at temperatures 274{356 K, two-dimensional 1 H{1 H NOESY spectra, and two-dimensional 1 H{13 C HMBC spectra for DNA sequences with lengths of 12, 14 and 16 nucleoti- des were acquired. The thermodynamic parameters of formation of the secondary structure in the samples were determined from the measured temperature depen- dencies. The hairpin formation in the samples was con rmed based on the NOESY spectra and the lack of dependency of the melting temperature on concentration. The observed difference of the secondary structure from B-DNA could serve as a possible explanation of the high a nity of SRF to CArG box. 1
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Structural properties and stability of DNA hairpins characterized by NMR spectroscopy
Socha, Ondřej ; Římal, Václav (advisor) ; Srb, Pavel (referee)
CArG box is a highly conservative DNA motif found in Serum Response Element (SRE), which regulates expression of c-fos gene. In this thesis, short oligodeoxynucleotides containing CArG box were measured using nuclear magnetic resonance spectroscopy to evaluate their ability to preferentially form hairpin over duplex. 1H spectra were measured at temperature range (274-360) K. We acquired thermodynamic parameters of the transition between hairpin and single strand by fitting the temperature- dependent chemical shifts. The hairpin structure of our oligonucleotide samples was confirmed by non- B-DNA patterns in NOESY spectra, absence of concentration dependence of melting, and other pieces of evidence. Thus, occurrence of unusual DNA conformation of CArG box in native DNA, potentially even cruciform is highly possible. This could explain the high affinity between SRE and its transcription factor. Powered by TCPDF (www.tcpdf.org)
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The presence and stability of DNA mini-hairpins
Bušková, Hana ; Římal, Václav (advisor) ; Srb, Pavel (referee)
The secondary structure of DNA is variable and depends on the sequence of nu- cleotides in a strand. While DNA can form duplexes, formations of three, four, or even a single strand have been observed in vivo and in vitro as well. In this thesis, we study the effect of small changes of oligonucleotide sequences on the stability of hairpins formed by DNA heptamers by 1 H nuclear magnetic resonance (NMR) spectroscopy. Suitable DNA sequences were selected based on symmetry rules and stability prediction by near- est neighbor model. Two-dimensional 1 H -1 H NOESY spectra were used to assign the 1 H resonances of aromatic hydrogens. Variable-temperature 1D spectra served for ob- taining melting curves, from which the thermodynamic properties of the hairpins were determined. The presence of hairpins in the solutions was confirmed by the character of the NOESY spectra, independence of melting temperature on oligonucleotide concen- tration, and comparison of competing melting-curve models of duplex and hairpin. Our results point out the importance of the order of the stem base pairs and contribute to the description of the extraordinary stability of DNA mini-hairpins. 1
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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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The thermodynamics of DNA-hairpin formation
Sgallová, Ráchel ; Římal, Václav (advisor) ; Profant, Václav (referee)
Serum response factor (SRF) is a transcription factor which binds to a highly conserved DNA sequence called the CArG box. According to the nucleotide sequence of CArG box it could form a hairpin structure or a cruciform. In this master thesis, the structure of the CArG box in a human gene c-fos was studied by nuclear magnetic resonance. 1 H spectra at temperatures 274{356 K, two-dimensional 1 H{1 H NOESY spectra, and two-dimensional 1 H{13 C HMBC spectra for DNA sequences with lengths of 12, 14 and 16 nucleoti- des were acquired. The thermodynamic parameters of formation of the secondary structure in the samples were determined from the measured temperature depen- dencies. The hairpin formation in the samples was con rmed based on the NOESY spectra and the lack of dependency of the melting temperature on concentration. The observed difference of the secondary structure from B-DNA could serve as a possible explanation of the high a nity of SRF to CArG box. 1
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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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Melting of a methylated CpG-containing DNA double helix
Bušková, Hana ; Římal, Václav (advisor) ; Hanyková, Lenka (referee)
The melting of a DNA double helix into two single strands is a crucial process for life on Earth. Previous experimental data on the self-complementary sequence CTTm5 CGAAG containing 5-methylcytosine suggests that the melting of an oligonucleotide might be a three-state, rather than two-state process. To assess this, 1 H NMR spectra were measured at varied concentrations of the oligonucleotide in temperatures between 274 and 366 K. Through analysis of the chemical shifts of aromatic hydrogens undergoing fast chemical exchange, we have determined that the most likely intermediate state between a double helix and a single stranded random coil is a structure consisting of two oligonucleotide molecules rather than a structured single strand.
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NMR Study of Oligonucleotide Structures
Římal, Václav ; Štěpánková, Helena (advisor) ; Spěváček, Jiří (referee) ; Sychrovský, Vladimír (referee)
Title: NMR Study of Oligonucleotide Structures Author: Václav Římal Department: Department of Low-Temperature Physics Supervisor: Prof. RNDr. Helena Štěpánková, CSc., Department of Low- Temperature Physics Abstract: The dynamics of nucleic acids plays a fundamental role in the interactions with proteins. Some processes are governed by changes in DNA stability and flexibility caused by sequence alterations or chemical modifications without substantial structural impact. We employed line-shape analysis of variable-temperature 1H NMR spectra to deeply investigate the melting transition of DNA structures. We observed a significant influence of the sequence beyond the nearest neighbours on chemical shifts and thermodynamics of the double helix. The CpG motif is especially sensitive to its distant surroundings and for a particular oligonucleotide it was even found to undergo an unexpected local conformational transition. A strong cooperativity in the duplex dis- ruption was witnessed by both the equilibrium and kinetic aspects as well as by distant effects of cytosine methylation. We propose a two-state melt- ing scheme also for a fragment of a transcription element proved here to fold as a hairpin with a six-membered loop. In addition, we describe the properties of self-assemblies of riboguanosine and...
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