Termodinamske značilnosti zvitja s citozini bogatih fragmentov DNA v i-motiv

Žerjav, Neža (author), Lah, Jurij (mentor)

Povezava protoniranega in neprotoniranega citozina s tremi vodikovimi vezmi je osnova strukture i-motivov. Ti lahko nastanejo iz nukleotidnih zaporedij, ki vsebujejo štiri zaporedne odseke citozinov in kakršna najdemo v telomernih, centromernih in regulatornih regijah genoma. Ker je za nastanek i-motivov potrebna protonacija enega od citozinov, i-motivi nastajajo pri pH ~ 5,0 in so pri fiziološkem pH pretežno nestabilni, zaradi česar je bil njihov obstoj in vivo dolgo časa vprašljiv. Kasnejše raziskave so pokazale njihovo prisotnost v celicah in nakazale možnost njihove vloge v procesih, kot so podvojevanje DNA, izražanje genov in podaljševanje telomer. Namen raziskave je bil določiti mehanizem in gonilne sile zvitja oligonukleotidov v i-motive na primeru različnih nukleotidnih zaporedij. Strukturne spremembe oligonukleotidov v odvisnosti od pH smo spremljali s CD-spektroskopijo. Po pričakovanju so se oligonukleotidi zvili v i-motiv pri pH ~ 5,0 z izjemo zaporedja 8bv6, ki se lahko nahaja v dveh konformacijah in je že pri fiziološkem pH v obliki i-motiva. pH smo spreminjali v območju med 7,0 in 4,5, preverili pa smo tudi reverzibilnost procesa z višanjem pH. Ugotovili smo, da je proces reverzibilen. Z UV-absorpcijsko spektroskopijo smo preverili reverzibilnost termične denaturacije i-motivov in ugotovili, da je proces ireverzibilen. S izotermno titracijsko kalorimetrijo smo spremljali toplotne učinke pri nižanju pH raztopine oligonukleotidov in posledičnem zvijanju le-teh v i-motive. Na podlagi rezultatov CD-spektroskopije in izotermne titracijske kalorimetrije smo na modelno neodvisen način izračunali termodinamske parametre tvorbe i-motiva. Z modelsko analizo podatkov smo opisali pH- in kalorimetrične titracijske krivulje za predpostavljeni mehanizem zvijanja. Izkazalo se je, da zvijanje poteka stopenjsko, preko vmesnih stanj z različnim številom vezanih protonov. Modelska analiza je opredelila termodinamske gonilne sile tvorbe i-motivov ter vmesnih stanj in njihovo populiranost v odvisnosti od pH.

• Language: Slovenian
• Keywords: DNA, i-motiv, termodinamika
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-170172
• COBISS.SI-ID: 242914563
• Publication date in RUL: 02.07.2025

Secondary language

• Language: English
• Title: Thermodynamic properties of folding of cytosine rich DNA fragments into i-motif

Abstract

The main feature of the i-motif structure are cytosine base pairs, each of them made of a protonated and an unprotonated cytosine connected with three hydrogen bonds. I-motifs can be formed from nucleotide sequences comprised of four stretches of cytosine-rich sequences interspersed with loop nucleotides. As the protonation of cytosines is crucial for formation of cytosine base pairs and it occurs at pH ~ 5,0, which is far below the physiological pH, it had long been speculated whether these structures do exist in vivo. Nevertheless, further studies confirmed their presence in living cells and brought up speculations about their role in processes such as DNA replication, gene expression and telomere lengthening. The aim of this research is to determine mechanism and driving forces of i-motif folding for various nucleotide sequences. Changes in conformation of the oligonucleotides depending on pH were determined using CD spectroscopy. As expected, i-motifs were mainly formed at pH ~ 5,0 except for the sequence 8bv6 which already at the physiological pH adopted one of its two possible i-motif conformations. Experiments were conducted in the pH range between 7,0 and 4,5. Reversibility of the process was confirmed by changing pH in the opposite direction. Furthermore, thermal denaturation was assessed with UV absorption spectroscopy. We concluded that the process is irreversible for all i-motifs. Isothermal titration calorimetry was used to measure heat effects accompanying proton binding and consequent folding of the nucleotides into i-motifs in the solution whose pH we were gradually lowering. The results of CD spectroscopy and isothermal titration calorimetry were used to determine thermodynamic parameters in a model-independent manner. Additionally, obtained pH and ITC titration curves were used for model analysis that provided insight into the folding mechanism. We assumed that folding is gradual, with protons binding singly. Based on the model we also determined driving forces of the i-motif formation and distribution of populations of partly folded i-motif species depending on the pH of the solution.

• Keywords: DNA, i-motif, thermodynamics