Book of Abstracts: Albany 2003
June 17-21 2003
Cisplatin and Self Splicing Activity of Group-I Intron Ribozyme
Group-I intron ribozyme is a unique class of RNA molecule that undergoes self-catalytic activity due to its unique folded structure and in the presence of guanosine co-factor and magnesium chloride. Several in vitro studies have shown that the folded structure of group-I intron ribozyme and its splicing can be a potential target site for the small molecules. Currently reports have shown that these type of intron are found to exist in 25s rRNA of C.albicans. Their presence in human pathogens and absence in humans suggests that the intron could act as an alternate target site compared to the known site of the pathogens. To investigate this we carried out a basic in vitro splicing assay using a gene fragment of 26s rRNA of Tetrahymena thermophila in the presence of cisplatin and transplatin and in vitro screening assay to analyze whether the agent used for our study will have any effect on 25s rRNA of C.albicans. The plasmid DNA pT7 was linearised containing the gene fragment and pre-rRNA was synthesized by run off transcription. Due to adequate amount of GTP and MgCl2 present in transcription mixture, we found the spliced out products on the gel concurrent with transcription, thus agents were added during transcription and the self-splicing was analyzed. This method also helps us in understanding whether cis-DDP has alternate affects on transcription besides splicing. The self-splicing was analyzed based upon the C-IVS formation which is a characteristic feature for the group-I intron in 26s rRNA of Tetrahymena thermophila. Interestingly we found that cis-DDP at 5μM and 40μM concentration failed to inhibit the self splicing process with 30 minutes of incubation. Increasing the time of incubation abolishes the spliced out products, such as c-IVS and L-IVS, which implies that cis-DDP though have fast leaving group needs longer incubation for the formation of number of free active Pt2+ to exert its biological activity on RNA. On the other hand with 5μM cis-DDP and 10mM MgCl2, a complete spliced outs especially C-IVS was analyzed suggest that, cis-DDP also possibly interacts at MgCl2 site besides other regions in intron. Further these agents were screened against clinical strains of C.albicans obtained from leprosy, hepatitis virus and throat swap. We used C.albicans as model since its harbors this intron in 25s rRNA and is highly prevalent in immunocomparised and cancer patients. Disk diffusion and growth curve analysis have shown that cis-DDP inhibit the growth of C.albicans at as low as 20 to 40μg concentration. While RNA extraction showed inhibition of both rRNA (25s and 18s) in C.albicans in the presence or absence of cis-DDP. Being a preliminary report we hereby show that i) Time dependent study showed loss of self splicing activity in vitro, ii) cis-DDP exhibits other biological activity besides its antitumorocidal property, iii) cis-DDP is not specific to 25s rRNA in bringing the inhibition, iv) cis-DDP showed higher affinity to all RNA?s besides its principle target.
Department of Genetics