Mao J R, Shimada M, Inouye S, Inouye M
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
J Biol Chem. 1995 Aug 25;270(34):19684-7. doi: 10.1074/jbc.270.34.19684.
Antisense technology has been widely used for regulating gene expression. Single-stranded RNA or DNA complementary to a target mRNA can inhibit the translation of the mRNA. Antisense RNA is produced in vivo, while antisense DNA is chemically synthesized as an oligonucleotide, which is extracellularly added to the cells. To maintain the effect of antisense DNA, a synthetic oligonucleotide has to be constantly added to the system. An advantage of antisense DNA over antisense RNA is that the target mRNA hybridized with the antisense DNA can be specifically digested by ribonuclease H. Here, we attempted to produce in vivo short single-stranded DNAs complementary to a specific mRNA. We demonstrate that such antisense oligodeoxyribonucleotide of a desired sequence can be produced in Escherichia coli using a retron, a bacterial retroelement, as a vector and that the antisense DNA thus produced in vivo can effectively inhibit the expression of a specific E. coli gene, such as the gene for the major outer membrane lipoprotein.
反义技术已被广泛用于调控基因表达。与靶标mRNA互补的单链RNA或DNA可抑制该mRNA的翻译。反义RNA在体内产生，而反义DNA则作为寡核苷酸进行化学合成，并在细胞外添加到细胞中。为维持反义DNA的作用，必须不断向系统中添加合成寡核苷酸。反义DNA相对于反义RNA的一个优势在于，与反义DNA杂交的靶标mRNA可被核糖核酸酶H特异性消化。在此，我们尝试在体内产生与特定mRNA互补的短单链DNA。我们证明，利用逆转录子（一种细菌反转录元件）作为载体，可在大肠杆菌中产生所需序列的此类反义寡脱氧核糖核苷酸，且在体内如此产生的反义DNA可有效抑制特定大肠杆菌基因的表达，如主要外膜脂蛋白基因。
