magistrsko delo
David Bezlaj (Author), Janez Plavec (Mentor), Jurij Lah (Thesis defence commission member), Miha Pavšič (Thesis defence commission member)

Abstract

Magistrsko delo je del študije usmerjene v proučevanje strukturnih lastnostih oligonukleotidov z različnim številom ponovitev d(ATTTC) s pomočjo NMR spektroskopije, CD spektroskopije, UV spektroskopije in nativne poliakrilamidne gelske elektroforeze. Te ponovitve se pojavljajo pri bolnikih spinocerebelarne ataksije tipa 37 in odrasle mioklonične epilepsije. Ponovitve d(ATTTC) vodijo do akumuliranja RNA v celici, ki veže RNA-vezne proteine in s tem preprečuje njihovo normalno delovanje. S pomočjo omenjenih spektroskopskih metod smo ugotovili, da so strukturne lastnosti proučevanih oligonukleotidov odvisne od prisotnosti Mg2+ in Ca2+ kationov. Osredotočili smo se na proučevanje oligonukleotida s tremi ponovitvami d(ATTTC), z delovnim imenom EP3, za katerega smo opazili najlepšo disperzijo 1H NMR signalov, kar je pomembno za detajlno strukturno karakterizacijo. V magistrskem delu so vključeni rezultati, ki so ključno pripomogli h kasnejšemu določanju modela DNA z visoko ločljivostjo. S pomočjo 13C- in 15N-izotopskega označevanja posameznih nukleotidov ter uporabo 2D NMR metod smo asignirali večino NOESY koleracijskih signalov. Ti so nam podali podroben vpogled v EP3 strukturo, ki se tvori v raztopini ob prisotnosti Mg2+ ionov. Ugotovili smo, da je struktura sestavljena iz dveh oligonukleotidov, ki sta med sabo povezana preko dveh C-C+ in dveh T-T baznih parov. Slednje predstavlja osrednji del dimerne strukture, v kateri vsak izmed oligonukleotidov deloma tvori lasnico oziroma strukturni motiv podoben lasnici, ki jo sestavljata dva A-T bazna para povezana s trinukleotidno zanko. Analiza posameznih oligonukleotidov z dvema ali več kot tremi ponovitvami d(ATTTC) je razkrila ravnotežje različnih DNA struktur. Detajlna strukturna karakterizacija posameznih zvrsti v ravnotežju je otežena predvsem zaradi širokih 1H NMR signalov, ki se med seboj prekrivajo. Kljub temu lahko na osnovi rezultatov CD in NMR spektroskopske analize sklepamo, da so strukture vseh preučevanih oligonukleotidov vsaj delno podobne in vsebujejo enake sekundarne strukturne motive.

Keywords

oligonukleotidi;ponovitve d(ATTTC);i-motiv;lasnica;strukturna analiza;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [D. Bezlaj]
UDC: 577.113:544.176(043.2)
COBISS: 171313667 Link will open in a new window
Views: 59
Downloads: 9
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Other data

Secondary language: English
Secondary title: Study of pentanucleotide repeat structures associated with spinocerebellar ataxia and epilepsy
Secondary abstract: This master’s thesis is part of the study focused on elucidating the structural properties of oligonucleotides with different numbers of d(ATTTC) repeats by means of NMR spectroscopy, CD spectroscopy and natural polyacrylamide gel electrophoresis. The d(ATTTC) repeats have been linked to spinocerebellar ataxia type 37 and adult myoclonic epilepsy. Transcription of d(ATTTC) repeats lead to the accumulation of RNA in the cell, which sequester RNA-binding proteins, thereby hindering their normal functions. With the help of the spectroscopic analysis we demonstrated that folding of all studied oligonucleotides depends on the presence of Mg2+ and Ca2+ cations in solution. We focused on studying the oligonucleotide with three d(ATTTC) repeats denominated ‘EP3’, which exhibits most amenable dispersion of 1H NMR signals in terms of detail structural analysis. The results of this master's work include characterization of EP3 that were crucial for the subsequent determination of its high-resolution structure. With the help of 13C- and 15N-isotopic labelling of individual nucleotides and the use of the 2D NMR methods, we assigned most of the observed NOE signals, which enabled detailed insights into the structure adopted by EP3 in the presence of Mg2+ cations. Furthermore, we found that the structure consists of two oligonucleotidesconnected via two C-C+ and two T-T base pairs. The latter represents the central part of the dimeric structure, in which each of the oligonucleotides comprises a segment that forms two A-T base pairs connected by a trinucleotide loop. Analysis of oligonucleotides with two or more than three d(ATTTC) repeats revealed an equilibrium of different DNA structures. Detailed structural characterization of individual species in equilibrium is aggravated mainly due to broad 1H NMR signals that are overlapped. Nevertheless, based on the results of CD and NMR spectroscopic analysis, we can conclude that the structural properties of all studied oligonucleotides are at least partially similar and contain the same secondary structural motifs.
Secondary keywords: NMR;oligonucleotide;DNA;d(ATTTC) repeats;i-motif;hairpin;Jedrska magnetna resonančna spektroskopija;Univerzitetna in visokošolska dela;
Type (COBISS): Master's thesis/paper
Study programme: 1000375
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Kemija
Pages: VIII f., 51 str.
ID: 19917022