Binding Site Design

Introduction

Actual ideas for therapeutic treatments of diseases like (???) include the use of virus for cell or tissue specific "drug" targeting. Where the delivery by the virus may cure the patient if targeted to the right cell, major problems are side effects coming up when mistargeted virus deliver their cargo to non-targeted cells. As the standard load of a virus is DNA and the pharmacological function results in expression of its information, our idea is to control the delivered DNA outcome. Therefore we want to use cell type specific micro RNA (miRNA) expression levels for activation of the therapeutic treatment.

As miRNAs are naturally used in cells to regulate translation, usage of the existing system for self-regulation sounds obvious. So we tried to create binding sites for miRNAs to target our constructs and either shut-down (off-targeting) or activate (on-targeting) gene expression in targeted tissues. In the off-targeting strategy the therapeutic gene is itself targeted for RNAi, so expression will persist only in non-targeted cells. In the on-targeting strategy, two different constructs built up an operator/repressor system (e.g. Tet-On/Off), where the regulator of the therapeutic gene, the repressor, is targeted for RNAi. Only cells which are able to suppress translation of the repressor will be affected from the therapeutic gene.

To achieve this, we used the random assembly PCR (raPCR) – method from iGEM2009-Heidelberg team and adopted it to our purposes. The differences: Sequences from 100 to 400 base pairs are requested. Oligos span over a whole binding site for a certain miRNA and shuffling occurs on the level of pattern creation. Several points need to be considered for setting up miRNA-binding site (miRBS) patterns:

• the right distance after the stop codon for efficient (or non-efficient) targeting of miRNAs
• distance and sequence between miRBS (spacer region)