Difference between revisions of "Part:BBa K337008"

Latest revision as of 02:29, 28 October 2010

hsa-miR-122 Binding site pattern (2BS) with randomised nt9-12

hsa-miR-122 binding site pattern, consisting of 2 binding sites (BS). The BS are seperated by sequences of 30 base pairs, which are inert in terms of miRNA binding. Additionally, half of the separation sequence is either in front or the end of the whole pattern, to be unconnected to standard restriction sites (BB-2, RFC12).

The binding sites were created by using oligos where the reverse complemented nucleotides 9-12 of the corresponding miRNA were randomised:

CAAACACCATNNNNACACTCCA

Sequence and Features

Characterization of synthetic microRNA binding site patterns against endogenous miR122

We used the miMeasure plasmid with the BB-2 standard to characterise binding sites for the endogenously expressed miR122. We cloned two different synthetic microRNA binding site patterns into our miMeasure construct plasmid: 3.1 BBa_K337008 and 1.3 BBa_K337000. As the binding site is inserted downstream of green fluorescent protein (EGFP), a regulation of EGFP expression is to be expected. miMeasure normalizes knockdown of EGFP to the unregulated blue fluorescent protein (EBFP2). By calculating the ratio of EGFP to EBFP2 we determined the knockdown percentage characteristic of the binding site patterns.

We tested these constructs in four different set-ups with three different cell lines:
• HeLa cells, which do not express miR122 endogenously
• HeLa cells cotransfected with miR122 to mimic endogenous expression
• HuH7 cells, which are liver cells known to express miR122
• HepG2 cells, liver cells known to express low amounts of miR122 (Douglas, 2010)

EGFP to EBFP2 ratios were measured with flow cytometry and microscopy. Measurement results for the four cell lines and the binding sites 3.1 BBa_K337008 and 1.3 BBa_K337000 are shown in Fig. 4 for all four cell line setups. As expected, no down regulation of EGFP expression was measured in HeLa cells due to the lack of miR122 therein. This serves as a control for the design of our binding sites as it is clear that they do not cross-react with other endogenously expressed miRNAs but are specific for miR122. Similarly, no down regulation was observed in HepG2 cells. The levels of miR122 expression in those cells are reportedly reduced by 99.5&, therefore also serving as a negative control. Both the construct 1.3 (containing two perfect binding sites with the extra 10bp spacer in between) and 3.1 (containing 3 perfect binding sites) lead to an increase in downregulation effect of EGFP in Huh7 cells and in HeLa cells cotransfected with miR122 in comparison to the single perfect binding site.

Figure 4: miMeasure in four different set-ups with three different cell lines The construct containing synthetic microRNA binding site patterns against endogenous miR122 transfected into different cell lines. The EGFP/EBFP2 ratio for the construct containing no binding site was set to one.

References

• Douglas D:Small Molecule Modifiers of MicroRNA miR-122 Function for the Treatment of Hepatitis C Virus Infection and Hepatocellular Carcinoma. JACS. 2010 May 15; 132(23):7976-81.