Weiler S, Ariatti M, Hawtrey A O
Department of Biochemistry, University of Durban-Westville, South Africa.
Biochem Pharmacol. 1988 Jun 15;37(12):2405-10. doi: 10.1016/0006-2952(88)90367-x.
The formation of transferrin-DNA complexes intended for ligand-directed transfection studies has been achieved through a hybridisation technique involving complementary homodeoxypolynucleotide chains attached to the participating protein and DNA species. Oligothymidylate residues (pT)n obtained by dicyclohexylcarbodiimide (CDI) polymerisation of thymidine-5'-monophosphate (5'-TMP) were activated to the 5'-imidazolides which on incubation with transferrin yielded the 5'linked phosphoramidates (pT)n-5'-transferrin. Homopolymeric chain extension of (pT)5-5'-transferrin by terminal transferase and dTTP at 30 degrees for 30 min yielded (pT) 300-5'-transferrin. Cleavage of the phosphoramide link in the polymer modified transferrin at 37 degrees was pronounced after 30 min although at 25 degrees hydrolysis was less than 5% after 4 hr. Poly(dT)-5'-transferrin readily hybridised with [3H]poly(dA)-tailed Pst 1 linearised pBR322 DNA. Resultant complexes were demonstrated by nitrocellulose filter binding and immunoprecipitation with anti-transferrin antibody. In contrast with poly(dT)-5'-transferrin, poly(dT)-5'-transferrin-poly(dA)-tailed pBR322 DNA complexes were stable at 37 degrees suggesting that annealing is followed by further stabilising interactions between the DNA and protein components.
用于配体导向转染研究的转铁蛋白 - DNA复合物的形成是通过一种杂交技术实现的，该技术涉及连接到参与的蛋白质和DNA分子上的互补同型脱氧多核苷酸链。通过胸腺嘧啶 - 5'-单磷酸（5'-TMP）的二环己基碳二亚胺（CDI）聚合获得的寡聚胸苷酸残基（pT）n被活化成5'-咪唑化物，其与转铁蛋白孵育后产生5'连接的磷酰胺酯（pT）n - 5'-转铁蛋白。在30℃下用末端转移酶和dTTP对（pT）5 - 5'-转铁蛋白进行同聚物链延伸30分钟，得到（pT）300 - 5'-转铁蛋白。在37℃下，聚合物修饰的转铁蛋白中的磷酰胺键在30分钟后明显断裂，尽管在25℃下4小时后水解小于5％。聚（dT） - 5'-转铁蛋白很容易与[3H]聚（dA）尾的Pst 1线性化pBR322 DNA杂交。通过硝酸纤维素滤膜结合和用抗转铁蛋白抗体进行免疫沉淀来证明所得复合物。与聚（dT） - 5'-转铁蛋白相比，聚（dT） - 5'-转铁蛋白 - 聚（dA）尾的pBR322 DNA复合物在37℃下是稳定的，这表明退火后DNA和蛋白质组分之间会有进一步的稳定相互作用。
