As the title of our project states, “DNA is not enough”. There are several upper-level regulation systems in superior organisms. Our main idea was using miRNA to tune down the expression of genes, having tissue-specific, exactly tuned gene therapy as objective.

Being able to tune the expression levels of a gene in mammalian cells is a great concept but it becomes an extremely powerful tool if we can predefine the construct that can bring this about. This was the inspiration behind creating a tool that can provide us a scheme that, if followed, can make it possible to control the expression of a gene in specific cell types.

Gene therapy is based on expression of a gene in a specific tissues. However, in addition to tissue specific expression of the gene, the ability to tune its expression level in mammalian cells is a very useful concept. Moreover, this becomes an extremely powerful tool if we can predefine a construct that can bring this about. This was the inspiration behind creating a tool that can provide us with a strategy which makes it possible to control the expression of a gene in specific cell types.

Our approach for realization of this objective was to provide functionalities that correspond closely to our experimental project. Additionally, we provide access to a strategy that guides the user through the cloning process and makes available the option to use characterized standard parts submitted by us in the MIT parts registry.

The miBricks project consists roughly of two ideas. The first being tuning of gene expression using shRNAs/miRNAs and the second being specific targeting of tissues. We intend to tune the expression of a gene by manipulating the binding affinity of a miRNA/shRNA towards the transcript of this gene which results in different expression levels. This is brought about by introducing different binding sites for the miRNA/shRNA in the 3'UTR of the gene. These binding sites differ from each other based on certain sequence based features. By computational methods, we can predict binding site that should be inserted so as to achieve a desired level of expression. Targeting of specific tissues is achieved by introducing binding sites for tissue specific endogenously expressed miRNA into the construct that brings about knockdown of a gene based on its presence or absence in the tissue.

miRNA binding site features

References!!!!!!!!!!!!!!!!!!!

mechanistic difference of perfect and miRNA binding site!!!!!!!!

miRNA are non-coding regulatory RNAs functioning as post-transcriptional gene silencers. After they are processed, they are usually 22 nucleotides long and they usually bind to the 3’UTR region of the mRNA (although they can also bind to the ORF or to the 5'UTR), forcing the mRNA into degradation or just repressing translation [Bartel, 2004].

In vegetal organisms, miRNA usually bind to the mRNA with extensive complementarity. In animals, interactions are more inexact, creating a lot of uncertainty in the in silico prediction of targets[?].

The seed of the miRNA is usually defined as the region centered in the nucleotides 2-7 in the 5’ end of the miRNA. For an efficient binding site extensive pairing is usually required between the seed and the seed match of the miRNA binding site. The miRNA seed and the seed match is the region where miRNA and mRNA are bound inside the AGO protein.

Outside the seed, the existence of supplemental pairing (at least 3 contiguous nucleotides and at best centered in nucleotides 13-16 of the miRNA) stabilizes the bound complex and increases the efficacy of the binding site.

Binding sites with a high local AU content around the binding site have proven to be more effective (possibly because of the destabilization of the mRNA secondary structure around the site). An arginine at position one of the binding site supposedly binds to a different protein of the RISC complex [Bartel, 2009], thus increasing the binding site efficiency significantly.

Binding site at the end or the beginning of the 3'UTR are more efficient than in the middle of the 3’UTR. Binding sites within the first 15 nucleotides after the stop codon are not effective, since this region of the mRNA is inside the ribosome when translations stops. Thus a bound RISC complex in this region will dissociate after every round of translation and can not follow it's usual mode of action [Grimson et al., 2007].

About the mechanistics of the repression, it has been shown that the repressive effect is much higher when the binding site for the miRNA is in the first 15 nt of the 3'UTR. This would match the hypothesis.... BLA BLA BLA

Targetscan Scores Jan ????

Figure 1: Interactions between two miRNAs and their binding sites. Examples to show different types of seeds.

- 6mer (abundance 21.5%): only the nucleotides 2-7 of the miRNA match with the mRNA.

- 7merA1 (abundance 15.1%): the nucleotides 2-7 match with the mRNA, and there is an adenine in position 1.

- 7merm8 (abundance 25%): the nucleotides 2-8 match with the mRNA.

- 8mer (abundance 19.8%): the nucleotides 2-8 match with the mRNA and there is an adenine in position 1.

The percentages of abundance are calculated among conserved mammalian sites for a highly conserved miRNA (Friedman et al. 2008)

Tissue specific miRNAs

A useful supplement to achieving tuned gene expression in cells is the ability to specifically target tissues where this should be carried out. Tissue targeting has, for quite some time, been an important field of research that has drawn much attention and is central to gene therapy. miBEAT[link] tool not only allows generation of binding sites that regulate the level of expression of a desired gene, but also employs strategies that help target the right tissue and exclude expression in others. This functionality is based on the principle of using tissue specific miRNA binding sites which can be introduced in the miTuner[link] construct easily.

A smart way to specifically target tissues is to exploit the presence and absence of tissue specific endogenous miRNA in the target to specifically express or exclude expression of the gene of interest in the target. We make use of two of strategies based on this principle, namely, on-targeting and off-targeting. The off-targeting concept has been applied previously [http://2010.igem.org/Team:Heidelberg/Modeling#References (Wenfang Shi et.al., 2008)] wherein an endogenous miRNA is selected such that it is not present in the target tissue (therefore the gene is expressed) and is present in all the off target tissues (knockdown of the transcripts). Thus the gene is specifically expressed in the target.

In addition to the off-targeting strategy, we designed a new strategy, the on-targeting. In this case, the miRNA is present in the target tissue and excluded from the off-targets. The binding site for this miRNA is present within the 3'UTR of a repressor gene (in our case TET/O2) construct. The operator for the repressor in turn precedes the gene of interest (miGene) in the miTuner or miMeasure constructs. Therefore in the presence of miRNA in the cell, repressor is degraded and miGene is expressed while in off-targets repressor is translated and represses the expression of miGene.

References