Thermodynamic properties of folding of cytosine rich DNA fragment from the promoter region of proto-oncogene c-myc

Molekula DNA je kot nosilka genetske informacije predmet številnih raziskav. Poleg dobro znane Watson-Crickove strukture DNA obstajajo tudi drugačne konformacije DNA, kot sta G-kvadrupleks in i-motiv. Pojavljata se v pomembnih regijah v genomu in sodelujeta pri procesih kot sta podvajanje in prepisovanje. Ker se pogosto tvorita v promotorskih regijah onkogenov, sta vedno bolj preučevana kot možni tarči za zdravila pri zdravljenju raka. V tem magistrskem delu smo se osredotočili na zvitje krajšega zaporedja DNA v i-motiv. Za njegovo tvorbo je v prvi vrsti potrebna protonacija citozina, kar vodi v nastanek C+-C para. Če je zaporedje bogato s citozini, se lahko tvori več takih parov in posledica je zvitje verige v urejeno strukturo. Predmet raziskave je bilo preučevanje zvitja in razvitja s citozini bogatega zaporedja DNA (5\u27-CCTTCCCCACCCTCCCCACCCTCA-3\u27) v i-motiv. Zaporedje najdemo v promotorski regiji človeškega protoonkogena c-myc, katerega preveliko izražanje je povezano z velikim številom rakavih obolenj. S kombinacijo spektroskopskih in kalorimetričnih meritev smo raziskovali termodinamiko zvitja in razvitja, ki smo ga dosegali z uravnavanjem pH in temperature. Izkazalo se je, da je zvitje, doseženo s spreminjanjem pH, reverzibilen proces, pri ohlajanju toplotno denaturiranega i-motiva pa se struktura ne povrne. Pri študiji smo ugotovili tudi, da vezanje protonov ni popolnoma kooperativno. Za spremembo konformacije je namreč dovolj že nastanek dveh ali treh od predvidenih petih citozinskih parov. Celotno entalpijo zvitja v i-motiv smo dobili z upoštevanjem dveh prispevkov in sicer vzpostavitev π–π interakcij med naloženimi bazami razvite verige ter nadaljnje zvitje v i-motiv. Proces je voden entalpijsko, entropijska sprememba in sprememba toplotne kapacitete pa sta negativni.The DNA molecule is a carrier of genetic information and a subject of extensive research. Besides the well-known Watson-Crick structure, there exist different DNA structures, for example G-quadruplexes and i-motifs. They appear in important regions in the genome and participate in processes such as replication and transcription. They are often found in the promoter regions of oncogenes and are extensively studied as possible drug targets in cancer treatment. In this thesis we focused on folding of a short DNA sequence into i-motif. For its formation, the protonation of cytosine and the emergence of C+-C pair is necessary. If the sequence is cytosine-rich, several such pairs may be formed and the result is folding of the chain into i-motif. The subject of this research is folding and unfolding of cytosine-rich sequence DNA (5\u27-CCTTCCCCACCCTCCCCACCCTCA-3\u27) to i-motif. The sequence is found in the promoter of human proto-oncogene c-myc, whose increased expression is associated with a large number of cancers. With a combination of spectroscopic and calorimetric measurements we investigated the thermodynamics of folding and unfolding, which was achieved by regulating pH and temperature. It turned out that folding, achieved by modifying pH is reversible process, while cooling the heat-denatured i-motif doesn’t lead to the initial i-motif structure. We found that binding of protons is not completely cooperative. For the observed change of conformation, it appears, that only two or three of the five expected cytosine pairs are needed for folding. The enthalpy of folding to i-motif was estimated by taking into account two contributions, namely the establishment of π-π interaction between stacked bases of unfolded chain and further folding to i-motif. Process is governed by negative change in enthalpy. Entropy and heat capacity change accompanying folding are also negative