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 (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 (Lewis et al. 2005). Therefore, a large proportion of the transcriptome may be subjected to miRNA-mediated control (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 (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 (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.
By engineering specific miRNA binding site patterns, the iGEM Team Heidelberg 2010 aims at gaining access to miRNA fingerprints present in every single cell. Special binding site patterns of miRNAs can be applied to specifically detect these fingerprints and adjust gene expression. Hence, those fingerprints can be used as reliable markers to distinguish between different cell types and cellular stages. Binding site patterns are therefore highly specific sensors, detecting the major cell regulators directly from within a cell. As the miRNA binding site patterns are modular in their structure; they will allow for the integrartion of different miRNA input signals into a single output that can be tuned over a high range. Just imagine a device that could distinguish the miRNA finger print of HIV infected cells from that of uninfected cells and would automatically kill all infected cells and thereby cure the patient even before the disease really started. Or imagine a device that could be used to selectively target every cell type in your body and would thereby enable effective cancer treatment and even safe gene and virotherapy. In short, miRNA binding site patterns will allow us to control the expression of every target gene of choice according to the unique miRNA fingerprint present in a certain cell type at a certain stage. Thus, we believe that miRNA binding site patterns will become a powerful tool not only in synthetic biology and basic life-science research, but also in the development of many different applications. In summary, we foresee miRNA binding site patterns as genetic controlling devices of the 21th century that will enable the specific control of any target gene both in vitro and in vivo.
In order to approach this topic systematically, the iGEM Team Heidelberg 2010 will apply and integrate principles of both evolution and rational design. By using a PCR-based random assembly method developed by the iGEM Team Heidelberg in 2009, we will create a large library of miRNA binding site patterns. Such a library is of a high value, as it contains binding site patterns detected by many different miRNA fingerprints with various specificities. For enabling a comparable and reliable characterization of the miRNA binding site patterns established, we will develop a standard reporter system for quantifying miRNA binding site pattern activity. That will be an important contribution to the growing miRNA community, as it will allow the standardized activity quantification and comparison of all engineered miRNA binding site patterns in the future. In order to facilitate the screening of our engineered binding site pattern library, we will develop a modular adeno associated virus (AAV) based screening method, that enables a quick selection of the best miRNA binding site pattern candidates, avoiding the expensive and time-consuming cloning, transfection and testing of each single binding site pattern from the library. Therefore, we will construct a standard AAV screening vector and develop a screening protocol that will allow an easy and robust screening of not only miRNA binding site pattern libraries, but also of other evolved libraries, like transcription factor and promoter libraries in the future.
We will use mathematical modeling in order to estimate the optimal starting conditions for our miRNA binding pattern libaray construction and screening. Accordingly we will screen miRNA databases in order to get information about binding site structures, specificities and relative position of binding sites on mRNAs and their influence on miRNA based mRNA targeting. By feeding the resulting information into a model, we hope to gain an understanding on miRNA binding site pattern structure and its relevance for miRNA-mRNA interaction. Furthermore, the screening of our self-engineered miRNA binding site pattern libraries will enable us to refine our model with new information. Thereby we will generate a feed-back loop between experimental data and our binding site pattern model. These theoretical indications should ease rational design of miRNA binding site patterns that would give an adjusted output (translational inhibition) according to a certain input (miRNA fingerprint) in the future.
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