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miRNAs are small(21-26nt in length) non coding RNA which modulate gene expression. They are known to target approximately 60% of the human transcriptome by imperfect complementary binding to the 3' UTRs of the mRNA. miRNAs are thus associated with a large repertoire of functions related to homeostatic and pathological states of the cell. The elucidation of miRNA function has faced several challenges since their identification since knockouts have been difficult to produce. As an alternative, several antisense oligonucleotide approaches were conceived and used for knockdown or silencing of miRNA. Chemical modifications (including LNA, 2' o-methyl, cholesterol addition etc) of these antisense oligonucleotides have been used to improve their stability and efficacy of silencing. However all of these approaches suffer from a common disadvantage of limited titration (dose dependence) and RNaseH dependent cleavage of resultant duplex.
We have tried to overcome this problem by using novel nucleic acid based enzymes (which we coined antagomirzymes) to selectively bind and cleave miRNA. Our initial work involved the testing and use of 10-23 DNAzymes having a central conserved motif and flanking regions complementary to miRNA which ensure the selectivity of the silencing enzyme to the miRNA of interest. We tested this class of enzymes against 2 miRNA - miR 372 and miR 373 in vitro and in cellulo whose results will be discussed in detail. Further chemical modifications of the DNA based antagomirzymes were explored for their effects of improving efficiency of cleavage. We have also demonstrated the use of hammerhead ribozymes to silence miRNA. These have a significant advantage over DNA based enzymes owing to their ability to be endogenously expressed. In this study we compared wild type and modified ribozymes for their efficiency to cleave miR-21 (a known oncomir) both in vitro and in cell culture models.
More recently, we have screened well known aminoglycosides for their ability to inhibit miRNA function using miR-21 as a model system. Our choice of these small molecules lies in the premise that aminoglycosides are known binders of RNA secondary structure. Of the 50 molecules we screened using a luciferase assay, we identified that streptomycin inhibits miR-21 function by direct interaction with pre-miR-21.
Results of such studies will comprehensively be discussed during the presentation.
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