7 research outputs found

    The triple helix: 50 years later, the outcome

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    Triplex-forming oligonucleotides constitute an interesting DNA sequence-specific tool that can be used to target cleaving or cross-linking agents, transcription factors or nucleases to a chosen site on the DNA. They are not only used as biotechnological tools but also to induce modifications on DNA with the aim to control gene expression, such as by site-directed mutagenesis or DNA recombination. Here, we report the state of art of the triplex-based anti-gene strategy 50 years after the discovery of such a structure, and we show the importance of the actual applications and the main challenges that we still have ahead of us

    Nové fluorescenční nukleotidy pro metabolické značení a konstrukci DNA sond

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    The aim of the thesis was to synthesize new nucleosides, nucleotides and the corresponding DNA probes bearing various fluorescent labels, which can be used for bioanalytical applications. In the first part of the thesis, 2'-deoxycytidine and the corresponding nucleoside triphosphate bearing tryptophan-based imidazolinone fluorophore were synthesized by Sonogashira cross-coupling reaction. The fluorophore showed sensitivity to pH and viscosity. Nucleotide was used for the construction of modified oligonucleotides (ON) and DNA by primer extension (PEX) or polymerase chain reaction (PCR). Labelled ON probe was used for sensing interaction with single-strand binding protein, which resulted in increased fluorescence intensity of modified ON. Next, thymidine and thymidine triphosphate labelled by bezylidene- tetrahydroxanthylium fluorophore were synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Fluorescence of the fluorophore is dependent on the polarity and viscosity of the environment. Incorporation of the modified nucleotide into DNA, by PEX or PCR, led to dramatic increase of the fluorescence presumably due to the interactions of the fluorophore in the major groove. Unfortunately, the modified nucleotide was not suitable for in cellulo imaging due to its cytotoxicity. The modified...Cílem této dizertační práce byla syntéza nových nukleosidů, nukleotidů a příslušných DNA sond nesoucích různé fluorescenční značky pro bioanalytické aplikace. V první části práce byl pomocí Sonogashiry cross-kapling reakce syntetizován 2'-deoxycytidin a jeho trifosfát nesoucí imidazolinový fluorofor na bázi tryptofanu. Daný fluorofor vykazoval citlivost na pH a viskozitu. Nukleotid byl použit pro konstrukci modifikovaných oligonukleotidů (ON) a DNA prodlužováním primeru (PEX) nebo polymerázovou řetězovou reakcí (PCR). Značená ON sonda byla použita pro snímání interakce se single-strand vazebným proteinem, což vedlo ke zvýšení intenzity fluorescence modifikovaného ON. Dále byl připraven thymidin a thymidin-5'-O-trifosfát, nesoucí benzyliden- tetrahydroxanthyliový fluorofor, pomocí mědí katalyzované azid-alkynové cykloadice (CuAAC). Fluorescence fluoroforu je závislá na polaritě a viskozitě prostředí. Inkorporace modifikovaného nukleotidu do DNA pomocí PEX nebo PCR vedlo k dramatickému zvýšení fluorescence, pravděpodobně díky interakcím fluoroforu ve velkém žlábku. Bohužel, modifikovaný nukleotid nebyl vhodný pro vizualizace v buňkách kvůli své cytotoxicitě. Modifikovaná dsDNA byla použita jako fluorescenční sonda pro snímání interakcí s malými molekulami a proteiny změnou fluorescence. Nakonec byl...Katedra organické chemieDepartment of Organic ChemistryFaculty of SciencePřírodovědecká fakult

    Development of Methodologies to Prepare Interstrand Cross-Links in Oligonucleotides

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    Cellular DNA is susceptible to damage by chemical agents that causes modifications which includes interstrand cross-links (ICL). ICLs covalently attach the complementary DNA strands, and interfere with replication and transcription by preventing strand separation. The deliberate formation of ICL in DNA by bi-functional alkylating chemotherapeutic agents leads to the death of cancer cells. Development of tumors resistant to these agents is a factor in the lack of response in some patients, with removal of ICL believed to play a role in resistance. In mammalian cells, the precise role of excision repair in eliminating ICL is not completely understood. For better understanding of the repair pathways involved in removing ICL damage, ICL DNA duplexes containing well-defined modified moieties are required to mimic the lesions induced by chemotherapeutic agents. This thesis describes two major approaches that have been investigated to synthesize ICL DNA. The first describes a method to prepare DNA duplexes containing cross-linked N3-butylene-N3 thymidines that enables the preparation of asymmetric nucleotide sequences around cross-linked sites. Protective groups for the 3’- and 5’-hydroxyl moieties were screened for compatibility of subsequent extensions from a cross-linked thymidine dimer incorporated in a support bound oligonucleotide by automated DNA synthesis. Two cross-linked dimer phosphoramidites were prepared, one with dimethoxytrityl (DMT) and allyloxycarbonyl (Alloc) protective groups at 5’-O positions and a 3’-O-t-butyldimethylsilyl (TBS) group which enabled the production of completely asymmetric ICL DNA duplexes in good yields. After coupling of the cross-linked phosphoramidite to a linear strand assembled on the solid support, the DMT group was cleaved on the synthesizer to allow for the synthesis of the second arm of the duplex. The Alloc group was then removed via an off-column strategy to expose the 5'-hydroxyl group to complete assembly of one strand of the duplex to form a "Y-shaped" intermediate. Final removal of a 3'-O-TBS group off column followed by coupling with deoxynucleoside 5'-phosphoramidites yielded ICL DNA duplexes containing completely asymmetric nucleotide composition around the cross-link site. The identity and composition of the ICL duplexes were confirmed by mass spectrometry (ESI-TOF) and enzymatic digestion. The synthesized ICL duplexes displayed characteristic features of a B-form duplex and had stabilities that were higher than those of the unmodified controls assessed by circular dichroism (CD) spectroscopy and UV thermal denaturation experiments. The second major project describes approaches to prepare a 7-deaza-2’-deoxyguanosine cross-linked dimer where the C7 atoms are attached by an alkylene linker. The chemical instability of alkylated N7 2’-deoxyguanosine (dG) represents a major challenge for preparing ICL DNA containing an alkylene linkage between the N7 atoms. The incorporation of a C7-alkylene cross-linked dimer of 7-deaza-2’-deoxyguanosine in DNA would allow for the preparation of a chemically stable ICL which mimic lesions formed by bifunctional alkylating agents (i.e. mechlorethamine and hepsulfam). Two synthetic methods were explored to prepare 7-deazaguanine (and other 7-deazapurines). These involved two cyclization strategies to prepare these molecules starting from a pyrimidine or a pyrrole to produce the purine. In both synthetic methods it was challenging to purify some of the intermediates. All intermediates in the synthetic method starting from the pyrimidine precursors to produce the 7-deazapurines were more stable while the production of the 7-deazapuines from the pyrroles resulted in higher yields. An attempt to produce a C7 cross-linked dimer of 7-deaza-2’-deoxyguanosine containing a heptamethylene linker is described. Starting from 7-deazaguanine, 7-iodo-7-deaza-2’-deoxyguanosine was prepared in good yield. This nucleoside was converted to 5’-O-DMT-7-iodo-7-deaza-2’-deoxyguanosine and the Sonogashira reaction used with 1,6-heptadiyne to introduce the heptamethylene linker. Unfortunately, multiple challenges were encountered with the dimerization and hydrogenation reactions which did not allow for the synthesis of the desired dimer for solid-phase synthesis of the ICL DNA

    Construction and functionalization of oligonucleotides

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    Oligonucleotides are significant tools in chemical biology and show wide applications. This thesis contains two projects concerning the construction of ligandosides and functionalization of oligonucleotides for formylcytosine detection. In the first project, a novel metal-base pair based on the pyrazole ligand was developed. The synthesis of the pyrazole ligandoside comprised the preparation of a protected base building block and a cuprate mediated C-glycosylation as the key step (Figure 1-1). The correct β-configuration of the nucleoside was confirmed by X-ray crystallography. The ligandoside precursor was incorporated into numerous oligonucleotides by automated DNA synthesis (Chapter 4.2). With a homo-pyrazole base pair inserted, duplex stability increased by 9°C after incooperation of one copper ion. The chelating performance depends on deprotonation of the phenol group of the ligandoside. Up to ten copper ions can be coordinated inside the duplex. Compared with the bridging salen base pair, the non-bridging pyrazole base pair shows a kinetical preference for complexation (Figure 1-2, Chapter 4.3). The pyrazole triphosphate is accepted by the Therminator polymerase which extends the primer. The unsatisfactory efficiency, however, hinders its application in PCR (Chapter 4.4.1). A duplex with five pyrazole-copper pairs was applied as a chiral catalyst in a model Diels-Alder reaction, which allowed to reach an ee value of 39% (Chapter 4.4.2). The novel ligandoside sheds light on how DNA may be used as a catalyst in organic reactions and enlightens further design and optimization of ligandosides. The second project is focused on developing a new fdC sequencing method based on an oligonucleotide probe connected to a hydroxylamine linker. The linker was selected using a combinational chemistry strategy. The most suitable linker at an n+4 position of the probe strand was known to react with the fdC in the target strand irreversibly. Because the probe and the target strand hybridized to form a duplex, the probe reacted with the target fdC with high positional specificity (Figure 1-3, Chapter 4.2). The reaction is limited to fdC and can tolerant single nucleotides polymorphisms in the target. Multiple fdC probes can be applied together. Enzymatic digestion and primer extension experiments were performed on the cross-linked oligonucleotide towards LC-MS and PCR detection (Chapter 4.3). After oxime formation, the duplex can be digested into dinucleotides but they cannot be detached. The probe strands hinders the Taq polymerase to pass through the target strand. A method for relative fdC quantification was developed using the described probe (Figure 1-4 Chapter 4.4). After crosslinking, the fdC probe was ligated to an adapter strand, wrapped in nanodroplets and replicated using PCR. The signals were counted and compared to a reference amplicon. 10-fold increase of fdC was observed at one position in an exon in Tdg-/- mES cells compared to Dnmt TKO cells, and 2-fold compared to Tdg+/- cells. The method can be applied to other targets of interest in order to track the dynamic change of the epigenetic fdC bases.Oligonukleotide sind wichtige Werkzeuge in der chemischen Biologie und erlauben breite Anwendungen. Diese Arbeit gliedert sich in zwei Projekte. Den Einbau von Metallo-Basen in DNA und die Funktionalisierung von Oligonukleotiden zur Detektion von 5-Formylcytosin. Im Zuge des ersten Projekts wird ein neues Metall-Basenpaar entwickelt, welches auf dem Pyrazolliganden basiert. Die Synthese des Pyrazolliganden erforderte die Darstellung eines geschützten Basenbausteins und eine Kuprat-vermittelte C-Glykosylierung als Schlüsselschritt (Abb.1-1). Die gewünschte β-Konfiguration des Nukleosids wurde durch eine Kristallstrukturanalyse bestätigt. Der Ligandosid- Vorläufer wurde in zahlreiche Oligonukleotide mittels automatisierter DNA-Synthese eingebaut (Kapitel 4.2). Liegt ein Homo-Pyrazol-Einzelbasenpaar in einem Strang vor, so erhöht die Zugabe von Cu2+ den Schmelzpunkt des Doppelstrangs um 9°C. Die Komplexierung hängt von der Deprotonierung des Phenols im Ligandosid ab. Duplexe mit 10 Pyrazol-Basenpaaren ermöglichte das Stapeln von bis zu zehn Kupferionen im Inneren eines Doppelstranges. Verglichen mit dem überbrückten Salen-Basenpaar, zeigte das nicht überbrückten Pyrazole-Basenpaar eine kinetische Präferenz zur Komplexierung (Abb.1-2, Kapitel 4.3). Die enzymatische DNA-Synthese erfolgt durch die Therminator Polymerase. Diese katalysiert die Polymerisation des Pyrazolnukleosid-triphosphats zu Polynukleotiden. Die unzureichende Effizienz steht weiteren Anwendungen in der PCR jedoch im Wege (Kapitel 4.4.1). Ein Duplexstrang mit fünf Pyrazol-Kupferpaaren wurde als chiraler Katalysator in einer Diels-Alder-Reaktion eingesetzt und erreicht einen ee-Wert von 39% (Kapitel 4.4.2). Der neue Ligandosid kann als DNA- Katalysator in organischen Reaktionen genutzt werden. Das zweite Projekt konzentriert sich auf die Entwicklung einer neuen Sequenzierungsmethode für fdC. Diese basiert auf einer Oligonukleotidsonde verbunden mit einem Hydroxylamin-Linker. Der Linker wurde durch kombinatorische Chemie entwickelt. Der am besten geeignete Linker an einer geeigneten Position auf dem Sondenstrang kann mit dem fdC des Zielstrangs irreversibel reagieren. Da die Sonde- und der Zielstrang zu einem Duplex hybridisieren, reagierte die Sonde mit dem erwünschten fdC mit Positionsspezifität (Abb.1-3, Kapitel 4.2). Die Reaktion ist spezifisch für fdC und toleriert Einzelnukleotid-Polymorphismus im Zielstrang. Mehrere fdC Sonden können gemeinsam angewendet werden. Enzymatische Hydrolyse und Primer-Extension-Experimente wurden an den vernetzten Oligonukleotiden durchgeführt. Dadurch konnten die Sonde durch LC-MS- und PCR-Methoden analysiert werden (Kapitel 4.3). Nach der Oximreaktion können die Doppelstränge in Dinukleotide verdaut aber nicht voneinander getrennt werden. Durch die kovalente Bindung zwischen den Sondensträngen wird die Taq-Polymerase gestoppt. Dies unterbindet die Vervielfältigung. Eine relative fdC Quantifizierungsmethode wurde mit dieser Sonde entwickelt (Abb.1-4, Kapitel 4.4). Nach der Vernetzung, wurden die fdC Sondestränge mit einem Adapterstrang ligiert, in Nanotröpfchen verteilt und repliziert. Die Signale in den Tröpfchen wurden gezählt und mit einem Referenzamplicon verglichen. So wurde in Tdg knokcout mES Zellen in Vergleich zu einem Dnmt Dreifachknockout die zehnfache Menge an fdC beobachtet und im Vergleich zu Tdg+/– Zellen die zweifache Menge. Das Verfahren kann auf eine Vielzahl von Zielen angewandet werden
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