Ahmed Anwar, Sharma Yagya D
Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Parasitol Res. 2008 Sep;103(4):751-63. doi: 10.1007/s00436-008-1036-y. Epub 2008 Jun 5.
Deoxyribonucleic acid (DNA) gyrase is an important enzyme that facilitates the movement of replication and transcription complexes through DNA by creating negative supercoils ahead of the complex. Its presence in Plasmodium falciparum is now established and considered a good drug target since it is absent in the human host. The sequence of P. falciparum gyrase A subunit was analyzed for its messenger ribonucleic acid (mRNA) folding as well as target accessibility for ribozymes. The four GUC triplet sites identified at 334, 491, 1907, and 2642 nucleotide positions of the Gyrase A mRNA were also accessible to oligos by RNase H assay. Site GUC491 was optimally accessible followed by GUC1907, GUC334, and GUC2642 sites. Ribozymes were produced against all these sites and tested for their in vitro transcript cleavage potentials where RZ491 showed the maximum cleavage rate. Therefore, this ribozyme (RZ491) was chemically synthesized albeit with modifications so as to make it resistant against ribonuclease attack. The modified ribozyme retained its cleavage potential and was able to inhibit the P. falciparum parasite growth up to 49.54% and 74.77% at 20 and 30 microM ribozyme concentrations, respectively, as compared to the untreated culture. However, up to 20% and 24.32% parasite growth inhibition was observed at the same ribozyme concentrations of 20 and 30 microM when compared with control ribozyme-treated cultures. This ribozyme as well as other targets identified here can be investigated further to develop the effective chemotherapeutic agents against malaria.
脱氧核糖核酸（DNA）促旋酶是一种重要的酶，它通过在复制和转录复合物之前产生负超螺旋，促进这些复合物在DNA上移动。目前已确定其在恶性疟原虫中存在，并且由于在人类宿主中不存在，被认为是一个良好的药物靶点。对恶性疟原虫促旋酶A亚基的序列进行了分析，以研究其信使核糖核酸（mRNA）折叠情况以及核酶的靶点可及性。通过核糖核酸酶H测定法还发现，在促旋酶A mRNA的334、491、1907和2642核苷酸位置处鉴定出的四个GUC三联体位点也可被寡核苷酸识别。GUC491位点的可及性最佳，其次是GUC1907、GUC334和GUC2642位点。针对所有这些位点制备了核酶，并测试了它们的体外转录物切割潜力，其中RZ491显示出最大的切割率。因此，尽管进行了修饰以使其对核糖核酸酶攻击具有抗性，但还是化学合成了这种核酶（RZ491）。修饰后的核酶保留了其切割潜力，与未处理的培养物相比，在20和30微摩尔核酶浓度下，分别能够抑制恶性疟原虫生长达49.54%和74.77%。然而，与对照核酶处理的培养物相比，在相同的20和30微摩尔核酶浓度下，观察到寄生虫生长抑制率分别高达20%和24.32%。这种核酶以及此处鉴定的其他靶点可以进一步研究，以开发针对疟疾的有效化疗药物。
