DEVELOPMENT OF NON-RADIOLABELLING METHOD OF NUCLEIC ACIDS

合成了黄素和5-萘磺酸的三(2-氨基乙基)胺类化合物作为新的核酸荧光标记剂。合成了可作为过氧化物酶模型化合物的聚乙二醇甲醚-海明偶合物的六甲二胺衍生物作为核酸的化学发光标记剂，而黄素衍生物在光存在下能降解DNA分子。将5-萘磺酸的三(2-氨基乙基)胺衍生物通过磷酰胺键成功地引入到15-聚体和21-聚体寡核苷酸的5‘端，并与标记的15-聚体及其互补DNA分子形成的双链相比具有更高的稳定性。这可能是由于5-萘磺酸部分上存在额外的自由氨基，可能与反DNA分子的磷酸部分形成离子键。标记的21-聚体具有部分自互补序列。因此，推测DNA在低温下形成对针环型二级结构，在高温下形成线型结构。为了探索利用荧光标记的DNA作为这种DNA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。研究发现，随着温度的变化，荧光强度和各向异性都发生了显著变化。结果表明，母体核酸的结构特征强烈地反映了标记剂的荧光特性。

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.