A Physical Study of Hairpin Type Double Helix and Triple Helix

• Main Author: 張治敏
• Other Authors: 靳宗玫
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/57812246879605820535

碩士 === 中國文化大學 === 應用化學研究所 === 91 === ABSTRACT In this thesis, a series of oligonucleotides, probe strands (5’-d-(AG)nT4(CT)n) and target strands (5’-d-(TC)n), were designed for the triple-helix formation in the PyPuPy motif. This systematical approach utilized Watson-Crick DNA hairpin probe strands to complex with the corresponding target strands through the formation of Hoogsteen hydrogen bonds. The formation and properties of hairpin triplexes were studied by UV thermal melting experiments and circular dichroism spectra. It was examined effects of the stem length of hairpin triplexes, the oligonucleotide concentration, the pH and the salts of buffer solutions. In the first part of the hairpin triplex study, the experimental data showed that the intermolecular triplexes could be formed as the length n≧3 in all conditions studied. The forming triplexes (n=4) at pH6.0 showed stabilities in a varity of buffer solutions: phosphate > cacodylate > MOPSO > BIS-TRIS > PIPES. The Tm1 values of the triplex (n=4) were 26.6, 22.4, 18.8, 17.2, and 12.4 ℃ in buffers of phosphate, cacodylate, MOPSO, BIS-TRIS, PIPES, respectively. However, the Tm2 values were 70.4, 71.2, 70.4, 70.4, and 71.2 ℃ in the above corresponding conditions. This indicated that the duplexes melted at almost the same temperature, but the melting temperature for dissocation of the Hoogsteen strand from the Watson-Crick duplex was related to the salt of a buffer solution. In addition, it showed a broad melting transition for the triplex (n=3-5) in the BIS-TRIS buffer. In the second part of the hairpin duplex study, it showed that the melting temperatures of the duplexes (n=2-4) were elevated as the stem length of the duplexes increased. In conditions of 0.02 M acetate buffer (pH6.0 and 4.5) or 0.02 M phosphate buffer (pH6.0), the melting temperature values of the duplexes (n=2-4) in oligonucleotide concentrations (2-100μM) were fluctuated in a small range (±3℃). This demonstrated that the formation of duplexes (n=2-4) is intramolecular. In conditions of 0.02 M acetate buffer (pH6.0) or phosphate buffer (pH6.0), the oligonucleotides (n=5-6) gave two UV themal transitions. The CD experimental data showed that the oligonucleotides could form intermolecular triplexes at the long stem length (n≧5).