Heidenreich O, Benseler F, Fahrenholz A, Eckstein F
Max-Planck-Institut für Experimentelle Medizin, Göttingen, Federal Republic of Germany.
J Biol Chem. 1994 Jan 21;269(3):2131-8.
The influence of chemical modifications on the catalytic activity and stability of a hammerhead ribozyme directed against the long terminal repeat RNA of the human immunodeficiency virus 1 was examined. Previous studies had shown that substitution of all pyrimidine nucleosides by their 2'-fluoro analogs led to an 8-fold decrease in catalytic efficiency in the cleavage reaction compared to the unmodified ribozyme (Heidenreich, O., and Eckstein, F. (1992) J. Biol. Chem. 267, 1904-1909). It is shown here that replacement of the 2'-fluoro-2'-deoxyuridines in the conserved region of this ribozyme, positions 4 and 7, by 2'-amino-2'-deoxyuridines fully restores catalytic activity of the ribozyme. Ribozymes containing these 2'-modifications show an increased stability against RNases present in fetal calf serum and in cell culture supernatant. The stability is increased further by the incorporation of four terminal phosphorothioates as protection against 3'-exonucleases, the degree of which depends on the secondary structure of the ribozyme. Such ribozymes are stable in undiluted fetal calf serum for at least 24 h. The results clearly demonstrate the potential to design stable ribozymes without any loss of catalytic activity.
研究了化学修饰对针对人类免疫缺陷病毒1长末端重复序列RNA的锤头状核酶催化活性和稳定性的影响。先前的研究表明，与未修饰的核酶相比，所有嘧啶核苷被其2'-氟类似物取代导致切割反应中的催化效率降低8倍（海登赖希，O.，和埃克斯坦，F.（1992年）《生物化学杂志》267，1904 - 1909）。本文表明，在该核酶保守区域（第4和7位）的2'-氟-2'-脱氧尿苷被2'-氨基-2'-脱氧尿苷取代后，完全恢复了核酶的催化活性。含有这些2'-修饰的核酶对胎牛血清和细胞培养上清液中存在的核糖核酸酶表现出更高的稳定性。通过掺入四个末端硫代磷酸酯作为对3'-外切核酸酶的保护，稳定性进一步提高，其程度取决于核酶的二级结构。这种核酶在未稀释的胎牛血清中至少稳定24小时。结果清楚地证明了设计出具有稳定活性且无催化活性损失的核酶的潜力。
