Termodinamske značilnosti pretvorbe dvoverižne DNA iz promotorske regije protoonkogena c-myc v G-kvadrupleks in i-motiv

Pikš, Katja (author), Lah, Jurij (mentor)

Znano je, da DNA poleg dvojne vijačnice lahko tvori tudi nekanonične strukture DNA, med katere spadata G-kvadrupleks in i-motiv. G-kvadrupleks se tvori iz zaporedja, bogatega z gvanini, i-motiv pa iz zaporedja, bogatega s citozini. Vse več raziskav potrjuje, da se ti dve strukturi lahko nahajata in vivo v človeških celicah. V človeškem genomu je omenjene strukture možno najti predvsem v telomernih zaporedjih, bogatih z gvanini, ter v promotorjih mnogih onkogenov. Tvorba G-kvadrupleksa v promotorski regiji protoonkogena c-myc sodeluje pri uravnavanju izražanja tega gena in tako predstavlja potencialno tarčo za zdravljenje raka. Komplementarna veriga, bogata s citozini, lahko tvori i-motiv. Namen našega dela je bil obravnavati pretvorbo oligonukleotidnega zaporedja iz promotorske regije c-myc med strukturami G-kvadrupleksa in i-motiva ter dvoverižno DNA v raztopini s spektroskopskimi tehnikami CD-spektroskopijo, UV-spektroskopijo ter fluorimetrijo. Potrdili smo tvorbo G-kvadrupleksa iz enoverižnega oligonukleotida Pu24G, tako pri pH 7,4 kot tudi pri pH 5,0 pri 100 mM koncentraciji kalijevih ionov. i-motiv iz oligonukleotida Pu24C se po pričakovanjih v raztopini tvori le pri pH 5,0, pri pH 7,4 pa ne nastane. Nastanek dvoverižne oblike DNA smo potrdili pri pH 7,4 z značilnim CD-spektrom in termičnim diferenčnim spektrom (TDS) za dupleks. Nastanek dupleksa pri pH 7,4 smo potrdili tudi s tehniko prenosa energije z resonanco fluorescence (FRET). Pri pH 5,0 je prisotna mešanica dupleksa, G-kvadrupleksa ter i-motiva, kar smo pokazali s pomočjo gelske elektroforeze. Pri spreminjanju pH mešanice se ravnotežna zmes vseh treh zvrsti v realnem času vzpostavi pri višanju pH od 4,5 do 7,4, pri nižanju pH od 7,4 do 4,5 pa je pretvorba kinetično omejena. Sestavo ravnotežne zmesi pri pH 5,0 smo lahko določili z analizo UV absorpcijskih spektrov in TDS. Pri pH 5,0 je ravnotežje pomaknjeno v smer nastanka G-kvadrupleksa in i-motiva, delež dvoverižne DNA je pri teh pogojih manjši.

• Language: Slovenian
• Keywords: DNA, c-myc, G-kvadrupleks, i-motiv
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-119688
• COBISS.SI-ID: 31248387
• Publication date in RUL: 10.09.2020

Secondary language

• Language: English
• Title: Thermodynamic properties of G-quadruplex, i-motif and duplex interconversion in DNA sequence from the promoter region of protooncogene c-myc

Abstract

Beside double helix it is well known that DNA can also form non-canonical structures, such as G-quadruplex and i-motif. G-quadruplexes and i-motifs can form in nucleic acids by sequences that are rich in guanines and cytosines, respectively. Both structures can appear in vivo in human cells. In the human genome, these structures are present in guanine-rich telomere sequences and in the promoters of many oncogenes. The formation of the G-quadruplex in the promoter region of the protooncogene c-myc is involved in regulation of expression of this gene and thus represents potential therapeutic target for cancer. Complementary strand rich in cytosines can form i-motif structure. The aim of our work was to study G-quadruplex, i-motif and duplex interconversion of oligonucleotide sequence from c-myc in solution using spectroscopic techniques CD spectroscopy, UV spectroscopy and fluorimetry. The formation of G-quadruplex from the single-stranded oligonucleotide Pu24G was confirmed at both pH 7.4 and pH 5.0 at 100 mM concentration of potassium ions. We also confirmed that i-motif in Pu24C oligonucleotide forms in solution at pH 5.0 only, whereas at pH 7.4 it does not form. Duplex forms at pH 7.4, which we confirmed by CD spectra, thermal difference spectra (TDS) and by fluorescence resonance energy transfer (FRET). Gel electrophoresis of mixture of duplex, G-quadruplex and i-motif was performed at pH 5,0 and we found that all three species are present. We examined the effect of changing pH between 7.4 and 5.0 on equilibrium of species. When changing the pH of the mixture from value 4.5 to 7.4, all three species are in equilibrium in real time. When lowering the pH from 7.4 to 4.5, the conversion does not happen in real time. We determined the composition of the equilibrium mixture at pH 5.0 using UV absorption spectra and TDS. At pH 5.0, the equilibrium shifts towards formation of G-quadruplex and i-motif, double-stranded DNA is present in smaller amount.

• Keywords: DNA, c-myc, G-quadruplex, i-motif