With the rising importance of small RNA molecules in gene therapy the identification and characterization of miRNAs and their binding sites become crucial for innovative applications. In order to exploit the miRNA ability to target and regulate specific genes, we constructed a measurement standard (see sidebar) not only to characterize existing miRNAs but also to validate potential synthetic miRNAs for a new therapeutic approach. The synthetic miRNAs we created lack endogenous targets and are thus applicable for gene regulation without any side effects. This openes new possiblities of precise expression tuning.
Our miMeasure constructs plasmid normalizes knockdown of the green fluorescent protein (EGFP) to the blue fluorescent protein (EBFP2). This allows an accurate study of binding site properties, since both fluorescent proteins are combined in the same construct and driven by the same bidirectional promoter. Another advantage is, that any desired binding site can be cloned easily into the miMeasure plasmid with the BB_2 standard. As the binding site is inserted downstream of EGFP, a regulation of EGFP expression is to be expected.
The percentage of knockdown of each modified binding site can be determined by the comparison of the ratio of EGFP to EBFP2 ratio compared to the ratio of the perfect binding site caused knock-down. The ratio is derived from a linear regression curve. Therefore the knock-down efficiency can be conducted by various basic methods e.g. plate reading, flow cytometry or microscopy.
Introduction
Micro RNAs regulate the translation of their target genes by preferably binding to regions in the 3’ UTR which are called miRNA binding sites (BS)(ref). This miRNA BS consists of a bp seed region at the 3'UTR that is perfectly matched to the miRNA, and surrounding regions that matched partially. The seed region is defined as being the minimal required basepairing at the 3’ end of the miRNA that can regulate the mRNA. Apart from the seed region, binding can be unspecific, creating bulges between miRNA and mRNA (fig). The position and properties of the bulges seem to play a role in miRNA binding and therefore knockdown efficiency (reviewed in Bartel et al., 2009).
Since we were going to use synthetic miRNA BS in our genetherapeutic approach, we had to find a way to study their effects in a standardized manner that would be comparable and reproducible.
One goal of the iGEM Team Heidelberg 2010 was to test the effects of changes in BS sequence and thereby characterize miRNA BS. To standardize our measurements of knockdown according to BS specificity, we had to come up with a new standard that is independent from the endogenous cell machinery. We decided to bring in synthetic miRNAs and engineer BS for them, simulating naturally occurring miRNAs and miRNA BS without having to worry about the effect of endogenous targets. Of course there are also differences that arise through the availability of the enzymes involved in the miRNA pathway that may differ slightly from cell to cell. Therefore, we also measured the knockdown achieved by the perfect binding site and set this as 100% knockdown efficiency. Ideally, the shRNA would be expressed stably in the cell line, but a uniform co-transfection also leads to an even distribution of shRNA into each cell.
The main idea of our measurement standard, miMeasure, was to express two nearly identical but discernable proteins, one of them tagged with a BS, the other one unregulated. These two reporters were expressed by a bidirectional CMV promoter to make sure their expression rate is identical. We used a destablilized version of GFP, dsEGFP by Clontech (ref) and a dsEBFP2 that was derived from the same sequence. Thus, we could make sure that both proteins exhibit the same synthesis and degradation properties, making them directly comparable. Hereby we can also neglect the difference between mRNA and protein knockdown and can take the fluorescence of the marker protein as a direct, linear output of mRNA knockdown. We included a BBB standard site into our plasmid, which allows to clone BS behind the GFP. If co-transfected with the corresponding shRNA, GFP will be downregulated, while BFP expression is maintained. The ratio of GFP to BFP expression can be used to conclude the knockdown efficiency (in percent, compared to perfect binding site=100% and no binding site=0%) of the BS. Having destabilized marker proteins with a turnover time of two hours enables us not only to avoid accumulation of marker proteins, which would make the knockdown harder to observe, but also to conduct time-lapse experiments. In the future, this could be for example a way to observe the activity patterns of endogenous miRNAs.
Results
Microscopy
Analysis of mutated/ randomized binding sites
We used microscopy analysis to determine the EGFP expression in relation to EBFP2. EBFP2 serves as a normalization for transfection efficiency. Nine miMeasure constructs with different binding sites were designed. The binding sites are either mutated at one site, or they contain randomly changed sites within a certain range. The construct representing the 100% knock-down is the perfect binding site, which is complementary to the synthetic miRNA miRsAg. The negative control represents 0% knock-down, since there is no binding site cloned into this miMeasure construct. M12 contains the perfect binding site. The GFP/BFP-ratio stand for the level of GFP-expression normalized to one copy per cell. When we compare the GFP/BFP-ratio between the constructs, there is a significant difference of GFP expression in the control (miMeasure without binding site) and the construct containing the perfect binding site for the cotransfected synthetic miRNA. The modified binding sites don't supress GFP-expression as much as the perfect one. So GFP expression is just in between, except for constructs M20 and M22.
Only the nucleotide 9 is mutated in M20. The change doesn't disturb the knock-down efficiency. Thus down-regulation of GFP is of similar extent as M12, which contains the perfect binding site. The seed region is altered in M22. Since the seed region is considered the most important site for knock-down efficiency, its change diminishes the knock-down capability of the binding site completely. So the GFP expression in this case is as high as the negative control, where no binding site was inserted into the miMeasure plasmid.
GFP/BFP ratio normalized by the negative control The data are generated by confocal microscopy of Hela cells, which were transfected with different miMeasure constructs (M12-M22) including the negative control (miMeasure construct without binding site). The negative control equals to 1.
Analysis of raPCR generated binding sites
The raPCR generates binding sites, which are aligned by chance with each other, whereby different random spacer regions are inserted in between. If perfect binding sites are aligned, this is predicted to give a stronger knock-down than a single one. If imperfect binding sites are aligned, it is also supposed to be stronger than a single one. In this way binding sites with different knock-down efficiencies can be generated.
This experiment shows, that the binding site pattern with 3 aligned perfect binding sites (miM-1.3-7) gives the strongest knock-down. Whereby the binding site pattern with two imperfect binding sites (miM-3.1-8) is weaker, but still stronger than the negative control. If one binding site is randomized from nucleotide 9-12 or 9-22 (miM-r12, miM-r22), it loses its capability of protein down-regulation.
Discussion
Methods
The fluorescence of GFP and BFP can be compared using different methods, for example automated fluorescence plate reader systems, flow cytometry or manual and automated fluorescence microscopy.
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