1992 Fiscal Year Final Research Report Summary
DEVELOPMENT OF NON-RADIOLABELLING METHOD OF NUCLEIC ACIDS
| Project/Area Number |
03804040
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
天然物有機化学
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| Research Institution | GUNMA UNIVERSITY |
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Principal Investigator |
SHINOZUKA Kazuo GUNMA UNIVERSITY FACULTY OF ENGINEERING ASSOCIATE PROFESSOR, 工学部, 助教授 (20206105)
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| Project Period (FY) |
1991 – 1992
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| Keywords | fluorescent labelling agent of DNA / flavin / 5-naphthalene sulfonic acid / poly(ethylene glycol methyl-ether)-Hemine conjugate / 過酸化水素分解酵素モデル |
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| Research Abstract |
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.
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