Study Of Folding And Unfolding Mechanism Of The I-MOTIF

Human telomeres are rich of cytosine (C) oligodeoxynucleotide DNA sequences. The C-rich olignucleotide sequences can forma four-stranded structure by the intercalation of two parallel duplexes containing hemiprotonated C·CH+in acidic environment, which is called i-motif. Most i-Motif structure was observed in the promoter region of oncogenes, such as VEGF, c-Myc and Rb promoter regions, making them becoming the attractive targets for gene-regulation process and for anti-cancer therapeutics. Therefore, the stability study of i-motif structure and exploration of folding mechanism has very important biological significance.In this thesis, our research object is the vertebrate telomeric sequence d (C3TA2)3C3. In order to have a good knowledge of the folding and unfolding mechanism of i-motif, we studied the influences of cytosine(C)to the stability and thermodynamics and kinetics of i-motif by the means of site-directed mutagenesis.First, cytosine of the telomeric sequence d (C3TA2)3C3was part mutated into adenine.we study the impacts of cytosine on the structural stability of the i-motif at the different positions using circular dichroism. The results showed that the structural stability of the mutant chains were poor and the the largest destabilization is observed for position C14.Second, we study the impacts of cytosine on the folding kinetics of the i-motif at the different positions using the stopped-flow technique (rapid mixing stopped-flow technique). The results show that the folding kinetics reaction rate of positions C2and C14mutations chain of are other mutations chain thousandth, indicating that these two mutation sites are key sites affecting the folding kinetics process of i-motif.Finally, we study the impacts of cytosine on the unfolding kinetics of the i-motif at the different positions using the method of Chemical denaturation by guanidine hydrochloride. The results showed that the structural stability of the positions C2and C14mutant chains were the poorest, indicating that these two mutation sites are key sites affecting unfolding kinetics of i-motif.Thus, we draw the conclusion that the positions C2and C14are key sites affecting the structural stability as well as thermodynamics and kinetics of i-motif, and playing an important role in the rate-limiting step in i-motif folding and unfolding process.