Vinayak Sumiti, Sharma Yagya D
Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India.
Oligonucleotides. 2007 Summer;17(2):189-200. doi: 10.1089/oli.2007.0075.
The nonmevalonate pathway of isoprenoid biosynthesis in the apicoplast of the human malaria parasite Plasmodium falciparum is distinct from the mevalonate-dependent pathway of humans and thus a good drug target. We describe here the hammerhead ribozyme based cleavage of the ispH (lytB) gene transcript involved in the last step of this nonmevalonate pathway. Using RNA folding program, three hammerhead ribozymes named as RZ(876), RZ(1260), and RZ(1331) were predicted against ispH (lytB) mRNA. Messenger walk screening (RNaseH) assay confirmed the target accessibility for these ribozymes. All three ribozymes cleaved the target RNA in vitro but RZ(876) exhibited the highest catalytic potential (62.92%). Therefore, RZ(876) was chemically synthesized with appropriate chemical modifications to protect it from nuclease attack while using it for in vitro parasite growth inhibition assay. This ribozyme RZ(876) was able to inhibit 87.36% parasite growth at 30 microM concentration compared to the untreated culture. However, an absolute inhibition of 29.41% was achieved compared to the control ribozyme (RZ(ctrl)). Nonetheless, the growth inhibition effect was found to be sequence-specific as indicated by the decreased level of ispH (lytB) transcript after ribozyme treatment. In conclusion, we have identified the ispH (lytB) as a potential target whose transcript can be cleaved by a ribozyme RZ(876).
人类疟原虫恶性疟原虫顶质体中类异戊二烯生物合成的非甲羟戊酸途径不同于人类的甲羟戊酸依赖途径，因此是一个很好的药物靶点。我们在此描述了基于锤头状核酶对参与该非甲羟戊酸途径最后一步的ispH（lytB）基因转录本的切割。使用RNA折叠程序，针对ispH（lytB）mRNA预测了三种锤头状核酶，分别命名为RZ(876)、RZ(1260)和RZ(1331)。信使步移筛选（RNaseH）分析证实了这些核酶对靶点的可及性。所有三种核酶均在体外切割了靶RNA，但RZ(876)表现出最高的催化潜力（62.92%）。因此，对RZ(876)进行了化学合成，并进行了适当的化学修饰，以保护其在用于体外寄生虫生长抑制试验时免受核酸酶攻击。与未处理的培养物相比，这种核酶RZ(876)在30 microM浓度下能够抑制87.36%的寄生虫生长。然而，与对照核酶（RZ(ctrl)）相比，实现了29.41%的绝对抑制。尽管如此，如核酶处理后ispH（lytB）转录本水平的降低所示，生长抑制作用具有序列特异性。总之，我们已确定ispH（lytB）是一个潜在靶点，其转录本可被核酶RZ(876)切割。
