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Development and characterization of light-producing deoxyribozymes
Švehlová, Kateřina ; Curtis, Edward Arthur (advisor) ; Höbartner, Claudia (referee) ; Renčiuk, Daniel (referee)
Light-producing protein enzymes such as luciferase play important roles in both applied and basic research. In this study, we used an in vitro selection to isolate deoxyribozymes that catalyze a chemiluminescent reaction by dephosphorylation of the commercial substrate CDP-Star. One of the most active variants, named Supernova, was further improved and characterized using a combination of random mutagenesis, in vitro reselection, high-throughput sequencing, comparative sequence analysis, and optimization of reaction conditions. Supernova produces light up to 6,500-fold more efficiently that the background reaction and folds into an unusual triple-helical structure. Moreover, we characterized in detail the buffer requirements including pH, the effect of various ions, substrate and Supernova concentrations, and the presence of crowding agents. Finally, we showed that Supernova can be turned into an allosteric sensor by rational design. We anticipate that this deoxyribozyme can be used as the signaling component in light-producing allosteric deoxyribozyme sensors that respond to a wide variety of stimuli and will complement existing methods that utilize radioactive, fluorescent, and colorimetric readouts.
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In Vitro Selection of Aptamers for Methionine Sulfoxide
Jureček, Matěj ; Míšek, Jiří (advisor) ; Bařinka, Cyril (referee)
Oxidation of methionine to methionine sulfoxide in proteins is considered one of important post-translational modifications of proteins. This modification can activate and also inhibit functions of many proteins and it is a part of regulation mechanisms of various (patho)physiological processes. For further research of the effects of methionine oxidation in proteins it would be very helpful to find its bioindicator. So far however, there has not been found any such antibody, nor any of its alternatives. This thesis was concerned with the search of ssDNA aptamer specific for methionine sulfoxide by the method of in vitro selection (SELEX). Several conditions for in vitro selection of methionine sulfoxide were tested in this diploma thesis. None of them led to the enrichment of the starting oligonucleotide pool and no selective aptamer for methionine sulfoxide has been found. Such results don't necessarily point to the impossibility of finding such aptamer, but the conventional methods used in this thesis weren't suitable for this task. In a control in vitro selection there has been found an enriched ssDNA pool for sulforhodamine B as a ligand. Sequencing of clones of this enriched pool has shown oligonucleotides with G-rich sequences, which is typical for already published aptamers for sulforhodamine B.
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Development of the new method for in vitro selection of DNA aptamers
Bláhová, Kamila ; Míšek, Jiří (advisor) ; Moserová, Michaela (referee)
Aptamers are short sequences of single-stranded DNA or RNA that are able to specifically bind various molecules (drugs, lipids, sugars, proteins, etc.). These aptamers are isolated from large libraries of random oligonucleotide sequences by SELEX (Systematic Evolution of Ligands by Component Enrichment) or in vitro selection. Despite the success of this method, in vitro selection often requires more than ten rounds of affinity selection as well as optimization of selection conditions. To increase the efficiency of aptamer selection, several methods have been developed that use an increase in the number of secondary structures in random oligonucleotide libraries. These methods, based on increasing the possibility of canonical base pairing in single-stranded oligonucleotides, increased the efficiency of the in vitro selection method. In this work, it was tested whether increasing the probability of occurrence of G-quadruplexes, as structural motifs in random sequences, will lead to increased efficiency of aptamer selection. Four single-stranded DNA libraries with different numbers of guanine (25 %, 35 %, 45 %, 55 %) in a random sequence were used. Streptavidin was chosen as the model molecule for selection, against which several aptamers that are not rich in guanine (G) have previously been selected....
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