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No previous studies have been performed to isolate and identify small regulatory RNA molecules including microRNA (miRNA) and double-stranded RNA (dsRNA) in the parasite B. malayi or in any other filarial nematodes. This thesis presents the design and implementation of a protocol to isolate and specifically clone small regulatory RNAs and their precursors in B. malayi that are cleaved by RNase-III-like nucleases such as Drosha and Dicer. We used B. malayi as the subject of our studies because its genome is best understood among the filarial parisites that infects humans. Using our protocol, we successfully cloned and sequenced twenty-five small RNA molecules ranging in size from (60-150) nucleotides (nts). We identified three potential miRNA and two putative small nuclear RNA (snRNA) candidates using computational approaches. Out of fourteen sequences that match protein-coding sequences in B. malayi, nine have the required minimal folding energy for miRNAS. These nine sequences could be precursors of miRNAs/miRNAs-like molecules. They can also be dsRNAs because they match the protein coding genes in Brugia's genome with almost 100% identity. Further investigation needs to be done to distinguish between the two. Five sequences that correspond protein-coding genes in Brugia's genome with 100% identity but do not have the stable folding energy of -20 kcal/mol are grouped together as endogenous dsRNA molecules. Our sequences were compared with sequences in the Entrez and miRBase databases. The secondary structures of sequences were generated using the Zuker algorithm with the web-based computational software Mfold. We calculated the Minimal Folding Energy Index (MFEI) of the RNA molecules using Zhang's formula for MFEI (Zhang et al., 2006). The MFEI calculated for ten C. elegans precursor miRNA molecules was in the range of 0.53-1.11. Two of the Brugia clones (ZA 023 and ZA 029) in this experiment have MFEI values of 0.58 and 0.60 (Table 3-4). These are in the range of MFEI calculated for C. elegans. The MFEI of the rest of the clones in miRNA category are in the range of 0.44-0.52 which is close to the lower bound of MFEI values found in C. elegans. This research lays the groundwork for further investigation of these small, regulatory RNA molecules in the parasite B. malayi. Uncovering the functions of these regulatory RNA molecules will provide us with some insight into some of the genes and proteins important for the development of the parasite. These genes and proteins will be the logical candidates for developing novel drugs to treat human lymphatic filariasis.
Aung, Zinmar, "Novel small regulatory RNA molecules in B. malayi : a route to discovering new potential drug targets for human lymphatic filariasis" (2008). Honors Project, Smith College, Northampton, MA.
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