Lee B L, Murakami A, Blake K R, Lin S B, Miller P S
Division of Biophysics, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205.
Biochemistry. 1988 May 3;27(9):3197-203. doi: 10.1021/bi00409a011.
Oligodeoxyribonucleoside methylphosphonates derivatized at the 5' end with 4'-(amino-alkyl)-4,5',8-trimethylpsoralen were prepared. The interaction of these psoralen-derivatized methylphosphonate oligomers with synthetic single-stranded DNAs 35 nucleotides in length was studied. Irradiation of a solution containing the 35-mer and its complementary methylphosphonate oligomer at 365 nm gave a cross-linked duplex produced by cycloaddition between the psoralen pyrone ring of the derivatized methylphosphonate oligomer and a thymine base of the DNA. Photoadduct formation could be reversed by irradiation at 254 nm. The rate and extent of cross-linking were dependent upon the length of the aminoalkyl linker between the trimethylpsoralen group and the 5' end of the methylphosphonate oligomer. Methylphosphonate oligomers derivatized with 4'-[[N-(2-aminoethyl)amino]methyl]- 4,5',8-trimethylpsoralen gave between 70% and 85% cross-linked product when irradiated for 20 min at 4 degrees C. Further irradiation did not increase cross-linking, and preirradiation of the psoralen-derivatized methylphosphonate oligomer at 365 nm reduced or prevented cross-linking. These results suggest that the methylphosphonate oligomers undergo both cross-linking and deactivation reactions when irradiated at 365 nm. The extent of cross-linking increased up to 10 microM oligomer concentration and dramatically decreased at temperatures above the estimated Tm of the methylphosphonate oligomer-DNA duplex. The cross-linking reaction was dependent upon the fidelity of base-pairing interactions between the methylphosphonate oligomers and the single-stranded DNA. Noncomplementary oligomers did not cross-link, and the extent of cross-linking of oligomers containing varying numbers of noncomplementary bases was greatly diminished or eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)
制备了在5'端用4'-(氨基烷基)-4,5',8-三甲基补骨脂素衍生化的寡脱氧核糖核苷甲基膦酸酯。研究了这些补骨脂素衍生化的甲基膦酸酯寡聚物与35个核苷酸长的合成单链DNA的相互作用。在365nm下照射含有35聚体及其互补甲基膦酸酯寡聚物的溶液，得到了由衍生化甲基膦酸酯寡聚物的补骨脂素吡喃环与DNA的胸腺嘧啶碱基之间的环加成产生的交联双链体。在254nm下照射可使光加合物形成逆转。交联的速率和程度取决于三甲基补骨脂素基团与甲基膦酸酯寡聚物5'端之间氨基烷基连接子的长度。用4'-[[N-(2-氨基乙基)氨基]甲基]-4,5',8-三甲基补骨脂素衍生化的甲基膦酸酯寡聚物在4℃下照射20分钟时产生70%至85%的交联产物。进一步照射不会增加交联，并且补骨脂素衍生化的甲基膦酸酯寡聚物在365nm下的预照射会减少或阻止交联。这些结果表明，甲基膦酸酯寡聚物在365nm下照射时会发生交联和失活反应。交联程度在寡聚物浓度高达10μM时增加，而在高于甲基膦酸酯寡聚物-DNA双链体估计的Tm的温度下显著降低。交联反应取决于甲基膦酸酯寡聚物与单链DNA之间碱基配对相互作用的保真度。非互补寡聚物不会交联，并且含有不同数量非互补碱基的寡聚物的交联程度大大降低或消除。（摘要截短于250字）
