Heidenreich O, Eckstein F
Max-Planck-Institut für Experimentelle Medizin, Göttingen, Federal Republic of Germany.
J Biol Chem. 1992 Jan 25;267(3):1904-9.
Three ribozymes targeted against different sites of the long terminal repeat RNA (LTR RNA) of human immunodeficiency virus type 1 cleaved a LTR RNA transcript 1,000 x less efficiently than corresponding short synthetic oligoribonucleotide substrates. Varying the stem lengths of the ribozyme resulted in changes of the catalytic efficiency. Almost no cleavage was observed for a ribozyme forming only 10 base pairs instead of 14 with the LTR RNA. Increasing the base pairs to 16 or elongation of the stem formed within the ribozyme revealed only small changes in kcat/Km. The influence of chemical modifications within the ribozyme on the cleavage of the LTR RNA was also examined. 2'-Fluorocytidine substitutions as well as four terminal phosphorothioate internucleotidic linkages influenced the catalytic efficiency of ribozymes only negligibly. However, substitution of uridine by 2'-fluorouridine resulted in a 5-fold decrease of kcat/Km. A ribozyme containing all these modifications revealed only a 7-fold lower catalytic efficiency but a markedly increased stability in cell culture supernatant. These results demonstrate that it is possible to increase the stability of ribozymes toward nucleases without a serious loss in catalytic efficiency.
三种靶向人类免疫缺陷病毒1型长末端重复RNA（LTR RNA）不同位点的核酶，切割LTR RNA转录本的效率比相应的短合成寡核糖核苷酸底物低1000倍。改变核酶的茎长度会导致催化效率的变化。对于与LTR RNA仅形成10个碱基对而非14个碱基对的核酶，几乎未观察到切割。将碱基对增加到16个或延长核酶内部形成的茎，仅显示出kcat/Km的微小变化。还研究了核酶内化学修饰对LTR RNA切割的影响。2'-氟胞苷取代以及四个末端硫代磷酸酯核苷酸间连接对核酶催化效率的影响可忽略不计。然而，用2'-氟尿苷取代尿苷导致kcat/Km降低5倍。含有所有这些修饰的核酶显示催化效率仅低7倍，但在细胞培养上清液中的稳定性明显增加。这些结果表明，有可能提高核酶对核酸酶的稳定性，而不会严重损失催化效率。
