DEVELOPMENT OF NON-RADIOLABELLING METHOD OF NUCLEIC ACIDS

合成了一种新的核酸荧光标记剂--黄素和5-萘磺酸的三（2-氨乙基）胺衍生物。合成了可作为过氧化物酶模型化合物的聚乙二醇甲醚-血红素共轭物的六亚甲基二胺衍生物作为核酸的荧光标记剂，但发现该黄素衍生物在光的存在下降解DNA分子。用氨基磷酸酯键将5-萘磺酸的三（2-氨乙基）胺衍生物成功地引入到15-mer和21-mer寡聚DNA的5 '端，并使标记的15-mer与其互补DNA分子形成的双链比未标记的15-mer与其互补DNA分子形成的双链稳定性提高。这可能是由于5-萘磺酸部分上存在额外的游离氨基，推测其与反向DNA分子的磷酸部分形成离子键，标记的21-mer具有部分自互补序列。因此，推测DNA在低温范围内形成pair-pin-loop型二级结构，在高温范围内形成线性型结构。为了研究利用荧光标记的DNA作为DNA的这种结构动力学的检测探针的可能性，分析了荧光强度和各向异性的温度依赖性。结果表明，随着温度的变化，荧光强度和各向异性都发生了显著的变化。结果表明，亲本核酸的结构特征强烈地反映了标记物的荧光特性。

Tris(2-aminoethyl)amine derivatives of flavin and 5-naphthalene sulfonic acid were synthesized as new fluorescent labelling agent of nucleic acids. Hexamethylene diamine derivative of poly(ethylene glycol methyl ether)-Hemine conjugate, which can be regarded as model compound of peroxidase, was synthesized as chemiluminescent labelling agent of nucleic acids.The above flavin derivative, however, was found to degrade DNA molecule in the presence of lights. Tris(2-aminoethyl)amine derivatives of 5-naphthalene sulfonic acid was successfully introduced to 5'-end of 15-mer and 21-mer oligoDNA through phosphoroamidate bond in satisfactory yields.The double strand formed by the labelled 15-mer and its complementary DNA molecule exhibited enhanced stability compared to the corresponding non-labelled 15-mer and its complementary DNA. This may be due to the presence of extra free amino group on 5-naphthalene sulfonic acid moiety which is presumably forming ionic bond with phosphate moiety of counter DNA molecule.The labelled 21-mer has partial self complementary sequence. Therefore, it is presumed that the DNA is forming pair-pin loop type of secondary structure at low temperature range and forming linear type of structure at high temperature range. To investigate the possibility utilizing the fluorescent labelled DNA for the detection probe of such structural dynamics of DNA, temperature dependency of fluorescent intensity and anisotoropy was analyzed. It was found that both the fluorescent intensity and anisotoropy change dramatically based on the change of temperature. The results indicate that the structural feature of parent nucleic acid strongly reflects the fluorescent characters of the labelling agent.