A Physical Study of Hairpin Type Triple Helix Formation － Effects of the Stem Length on the Stability of a Triple Helix

• Main Author: 黃鴻文
• Other Authors: 靳宗玫
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/52621264817692402522

碩士 === 中國文化大學 === 應用化學研究所 === 90 === Intermolecular triple helices have been of great scientific interest due to their possible therapeutic uses. In this report, we have developed an approach for targeting pyrimidine DNA and RNA single strands by the triple helix formation in the PyPuPy motif. This systematical approach used Watson-Crick DNA hairpin probes (5'-d-(AG)nT4(CT)n, n = 2-5) to complex with the corresponding target pyrimidine strands (5'-d-(TC)n, n = 2-5) through the formation of Hoogsteen hydrogen bonds. The formation of DNA hairpin type triplexes was monitored by UV thermal denaturation analysis and circular dichroism spectroscopy. Effects of the stem length (of the hairpin triplexes), the temperature, the solution pH (pH’s 4.5 and 6.0), the nature of buffer salts (phosphate and acetate), and cations (Mg2+ and spermine) on the stability of a complex were examined. The experimental results showed that the short intermolecular triplexes could be formed as the stem length n > 2 in all conditions studied. The resulting triplex (n = 3-5) gave two UV thermal transitions at pH 6.0 and single cooperative transition at pH 4.5. This indicated that the complexes formed stable triplexes at pH 4.5 with considerable stability as high as the corresponding hairpin duplexes in the same conditions. In addition, the phosphate buffer facilitated the triplex formation better than the acetate buffer. This reflects on the UV melting temperatures. In the case of the stem length n = 5, the Hoogsteen domain dissociation of the triplex showed a Tm value of 47 ℃ in the phosphate buffer solution of pH 6.0, while 34 ℃ in the acetate buffer solution of the same pH. On the other hand, the duplex AG5 melted at the same temperature in both buffers. Obviously, the stability of a triplex, would be affected significantey by the salt of a buffer solution.