Micro RNAs (miRNAs), a class of abundant small noncoding RNAs, are key regulators in all kinds of organisms ranging from viruses to mammals. By binding to target sequences most commonly found in the 3' untranslated region (UTR) of the mRNA, miRNAs inhibit the translation of their target mRNAs and thereby adjust the expression of many proteins related to the miRNA expression in a cell [http://2010.igem.org/Team:Heidelberg/Project/Introduction#References (Brenecke et al. 2005)]. The importance of miRNA-mediated gene regulation is impressively reflected by the fact that roughly 1% of the human genome codes for miRNAs which target 20-25% of all protein coding genes [http://2010.igem.org/Team:Heidelberg/Project/Introduction#References (Lewis et al. 2005)]. Therefore, a large proportion of the transcriptome may be subjected to miRNA-mediated control [http://2010.igem.org/Team:Heidelberg/Project/Introduction#References (Lau et al. 2001)]. The broad regulatory scope of miRNAs underlines their key roles in a wide range of biological processes including proliferation, apoptosis, hematopoeisis and oncogenesis [http://2010.igem.org/Team:Heidelberg/Project/Introduction#References (Bushati and Cohen, 2007)]. The expression patterns of miRNAs in different cell types, tissues and developmental stages of a cell vary highly, but remain relatively constant within a certain single cell type in a certain stage [http://2010.igem.org/Team:Heidelberg/Project/Introduction#References (Brown et al. 2006 , Gangaragu and Lin 2009)]. Thus, we perceive each cell type and cell state to represent a distinct inner-cellular miRNA expression pattern we refer to as 'miRNA finger print', which is distinguishable from that of any other cell type.
The special properties of miRNA binding sites and the inhibiting character of miRNAs are excellent devices to be exploited for gene therapy. Not only the fine-tuning capabilities of varying miRNA binding sites, but also the possibility to distinguish different cell types by their miRNA expression profile make synthetic miRNA binding sites an excellent tool to regulate translation in vitro and in vivo.
The main focus of our project were the creation of binding site libraries for synthetic as well as endogenous miRNAs by randomized and rational design methods. We established a protocol for PCR-based random assembly of binding site fragments and created a program for the in silico design of miRNA binding sites.
For enabling a comparable and reliable characterization of the miRNA binding sites, we developed a standard reporter system for quantifying miRNA binding site pattern activity called miMeasure. This will allow the standardized activity quantification and comparison of all engineered miRNA binding site patterns in the future.
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