MicroRNAs (miRNAs) are endogenous 22-nt long non-coding RNAs that regulate gene expression post-transcriptionally in a diversity of organisms (Bartel, 2004). Although the understanding of miRNA biological functions is progressing remarkably, the exact mechanisms by which miRNAs regulate gene expression are still not unambiguously defined. However, it is commonly believed that miRNAs trigger target mRNA regulation by binding to 3’UTRs (Chekulaeva & Filipowicz, 2009). The discovery of the first miRNA (lin-4) revealed sequence complementarity to multiple conserved sites in the 3’UTR of the lin-14 mRNA (Lee et al., 1993; Wightman et al., 1993). Exact principles of gene expression knockdown mediated by miRNA are still in debate (Eulalio et al., 2008).
Binding site properties have an essential impact on miRNA-mRNA interaction [figure, short explanations on seed regions, flanking regions, spacers, mismatches and resulting bulges]. Some functionally important parts of miRNAs have been described in literature, such as the seed region (Grimson et al., 2007; Bartel, 2009). It is defined as a region of seven nucleotides length that shows perfect pairing between the miRNA and its target sequence. The seed usually consists of the nucleotides on position 2-8 of a miRNA binding sites in the 5'UTR of the mRNA. Based on this simple principle, we randomized our miRNA binding sites between nucleotide 9 - 12 or 9 - 22 in the so called flanking region. Alternatively, we tried rational exchanges of nucleotides to see how they effect binding of the miRNA to its target mRNA. A combination of random and rational design of binding sites could thereby help to achieve a narrow range of resulting knockdown. Hence, we wrote a programm - the miBS designer - for in silico construction of synthetic miRNA binding sites. The experimental applicability is still limited by redundant target sites and various miRNA expression patterns within the cells. This hampers distinct expression levels of the gene of interest (GOI) fused to the miRNA binding site. We (want to overcome) these limitations in vitro and in vivo with our synthetic miRNA Kit.
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