Assembly of Novel DNA Cycles with Rigid Tetrahedral Linkers
Assembly of Novel DNA Cycles with Rigid Tetrahedral Linkers
复制标题
用刚性四面体接头组装新型 DNA 循环
DOI：
10.1002/anie.199701111
复制
发表时间：
1997
影响因子：
--
通讯作者：
D. Bergstrom
中科院分区：
文献类型：
--
作者：
Jufang Shi;D. Bergstrom
将含有寡核苷酸缀合物4a-4c的溶液用浓NH3、OH处理以除去保护基团，然后用H、O稀释并冻干。粗产物直接通过聚丙烯酰胺凝胶电泳(PAGE)纯化。通常，主条带（参见实验部分的检测方法）对应于所需的缀合物；对于双臂 12 聚体 (24 nt)，该条带略低于二甲苯基氰醇（XC，20% 变性 PAGE，相当于大约 28 聚体），而双臂 14 聚体和 16 聚体（28 和 32 nt）的条带则高于 XC。变性凝胶电泳后，切割所需条带并在 H、O 中洗脱，通过 C18 反相色谱或过滤脱盐，得到纯产物。通过基质辅助激光解吸电离 (MALDI) 质谱法确认了缀合物的结构。 ['~] 非变性 PAGE 用于表征寡核苷酸缀合物的杂交产物。纯化的双臂自互补寡核苷酸缀合物4a-4c形成明显的条带“阶梯”，其大小从24个碱基对到98个碱基对连续变化。缀合物 4b 由 12 聚体自互补寡核苷酸片段组成，其上附加了两个额外的胸腺嘧啶 (T) 残基，给出了相对干净的“阶梯”，其中至少有 8 个条带清晰可辨（图 3）。将 4c 与
The solutions containing oligonucleotide conjugates 4a-4c were treated with concentrated NH, OH to remove the protecting groups, and then diluted with H, O and Iyophilized. The crude products were purified directly by polyacrylamide geI electrophoresis (PAGE). Typically the major band (see Experimental Section for method of detection) corresponded to the desired conjugates; for a two-arm 12-mer (24 nt) that band was just below that of xylyl-cyanol (XC, 20% denatured PAGE, corresponding to approximately a 28-mer) while bands of the two-arm 14-and 16-mers (28 and 32 nt) ran above that of XC. After denaturing gel electrophoresis, the desired bands were cut and eluted in H, O, desalted either by C18 reverse-phase chromatography or filtration to yield pure products. The structures of the conjugates were confirmed by matrix-assisted laser desorption ionization (MALDI) mass spe~ trometry.[’~] Native PAGE was used to characterize the hybridized products from the oligonucleotide conjugates. The purified two-arm self-complementary oligonucleotide conjugates 4a-4c formed distinct “ladders” of bands, which ranged in size continuously from 24 base-pairs to 98 base-pairs. Conjugate 4b, which consists of a 12-mer selfcomplementary oligonucleotide segment to which two additional thymine (T) residues were attached, gave a relatively clean “ladder”, in which at least eight bands are clearly discernible (Figure 3). Conjugate 4c with