Študij predzvitih struktur G-kvadrupleksov človeškega telomernega zaporedja z uporabo jedrske magnetne resonance : enovit magistrski študij farmacija

Frelih, Tjaša (author), Anderluh, Marko (mentor), Šket, Primož (comentor)

Poleg dobro poznane desnosučne dvojne vijačnice lahko DNA tvori še druge strukture višjega reda, kot so G-kvadrupleksi. To so štirivijačne strukture, ki jih lahko tvorijo z gvanini bogata zaporedja. Za njihovo tvorbo so ključnega pomena kationi (K+ in Na+). O tem, kakšna je struktura z gvanini bogatih zaporedij pred dodatkom kationov, ni veliko znanega, zato smo se v okviru magistrskega dela osredotočili na predzvite strukture G-kvadrupleksov. Proučevali smo strukturne lastnosti človeškega telomernega zaporedja v vodni raztopini brez dodanih kationov z uporabo jedrske magnetne resonance in ostalih spektroskopskih metod. Ugotovili smo, da zaporedje tvori stabilno predzvito strukturo. Optimalni pogoji za proučevanje zaporedja so bili pH-vrednost 5 in 5 ºC. S pomočjo delnega izotopskega 15N, 13C označevanja posameznih nukleotidov v zaporedju smo nedvoumno asignirali signale v imino območju 1D 1H NMR spektra. NOE kontakti v imino območju 2D NOESY NMR spektra so nam pomagali pri določitvi topologije predzvite strukture, saj nam dajo informacijo o tem, kateri protoni so si v prostoru blizu skupaj. Proučevali smo dva oligonukleotida, ki se razlikujeta le v dveh timinih na 3'-koncu. Prvi je bil dolg 23 nukleotidov - d(TAGGG(TTAGGG)3), drugi pa 25 nukleotidov - d(TAGGG(TTAGGG)3TT). Oba oligonukleotida sta tvorila zelo podobno predzvito strukturo, ki je bila zaradi nižje pH-vrednosti protonirana. V predpostavljeni topologiji zadnji G-trakt, ki ga sestavljajo G21, G22 in G23, ni imel velike vloge pri tvorbi predzvite strukture. S pomočjo modifikacij zaporedja, smo določili najkrajše zaporedje, ki je potrebno za tvorbo predzvite strukture - d(TAGGG(TTAGGG)2TTA). S spreminjanjem pH-vrednosti med 5 in 7 smo spreminjali tudi ravnotežje med protonirano in neprotonirano obliko. Pri pH-vrednosti 7 smo opazili nastanek stabilne neprotonirane predzvite strukture. Tvorba G-kvadrupleksov poteka preko različnih intermediatov, ki so na splošno nestabilni in jih je zato težko odkriti. Uspelo nam je določiti enega od stabilnih intermediatov človeškega telomernega zaporedja, kar daje nov pogled na mehanizme zvijanja G-kvadrupleksov. Zaporedje se v vodni raztopini ne nahaja kot naključni klobčič, ampak je že organizirano in ob dodatku kationov hitro nastane G-kvadrupleks. Predzvite strukture lahko predstavljajo aktivne tarče v razvoju novih zdravil, zato poznavanje njihove strukture ponuja novo možnost za zdravljenje rakavih obolenj.

• Language: Slovenian
• Keywords: G-kvadruplks predzvita struktura človeško telomerno zaporedje kationi nuklearna magnetna resonanca
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [T. Frelih]
• Year: 2017
• Number of pages: IV, 63 f.
• PID: 20.500.12556/RUL-120500
• UDC: 543.429.23:577(043.3)
• COBISS.SI-ID: 4413041
• Publication date in RUL: 21.09.2020

Secondary language

• Language: English
• Title: Study of G-quadroplex pre-folded structures of human telomere sequence using nuclear magnetic resonance

Abstract

Besides well-known right-handed double helix, DNA can also form other higher-order structures, such as G-quadruplexes. G-quadruplexes are formed by four-stranded guanine-rich DNA sequences. Their formation requires presence of cations such as K+ and Na+. It is very little known about structures of guanine-rich sequences without cations. In the current study we focused on G-quadruplex pre-folded structures. Using nuclear magnetic resonance and other spectroscopic methods we studied structural properties of human telomere sequence in the aqueous solution without added cations. We have discovered that human telomere sequence form a stable pre-folded structure and optimal experimental conditions were pH 5 and 5 ºC. Unambiguous assignment of imino region of 1D 1H NMR spectrum was achieved by partly site-specific isotopically 15N, 13C labelled oligonucleotides. Imino-imino NOE connectivities in 2D NOESY NMR spectra helped us to determine the topology of pre-folded structure formed by human telomere sequence, because they give us the information about the protons that are close one to another. We studied two oligonucleotides that differ only in two thymines at 3’-end. The first one was 23 nucleotides long - d(TAGGG(TTAGGG)3) and the second one was 25 nucleotides long - d(TAGGG(TTAGGG)3TT), but both formed similar pre-folded structure. Due to the lower pH it was protonated. In the proposed topology the last G-tract consisting of G21, G22 and G23 residues did not play an important role in the formation of pre-folded structure. By sequence modifications we determined the shortest sequence required to form pre-folded structure to be d(TAGGG(TTAGGG)2TTA). By varying the pH between 5 and 7, the equilibrium between protonated and unprotonated form changed. At pH 7 the formation of stable unprotonated pre-folded structure was observed. The formation of G-quadruplexes proceeds via various intermediates, which are in general unstable and therefore difficult to detect. We managed to determine one stable intermediat of human telomere sequence which gives a new prospective on the mechanisms of G-quadruplex folding. In the solution the sequence is not a random coil, but it is pre-organised and when the cations are added it quickly folds into G-quadruplex. The pre-folded structures can be used as targets in the development of new medicines. This offers a new possibility for anticancer therapy.

• Keywords: G-quadruplex pre-folded structure human telomere sequence NMR spectroscopy cations