Team:MIT/Experiments/miRNA

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  Circuit Design Clinical Applications
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What can we learn from miRNA?

miRNA are small units of RNA that play a regular role in gene regulation. miRNA binds and cleaves messenger RNA, inhibiting gene expression. There are hundreds of miRNA, and their profile and activity vary between different cell types.




This makes it is possible to tell the difference between cells from the uterus and liver based on miRNA profiles. Furthermore, cells from the uterus of women with endometriosis have been found to have dysregulated miRNA in comparison to cells from the uterus of women who do not have endometriosis().

Found in all mammalian cells


Unique to cell type


Unique to cell states

How can our circuit sense miRNA?

Our circuit utilizes the natural function of miRNA to produce a differential output depending on whether the cell has dysregulated miRNA. This is achieved by attaching 4 tandem sites complementary to the affected miRNA following a gene of interest.

Are our target sites sensitive to miRNA levels?

These 4 tandem sites for miRNA binding were tested by coding for them distal to the gene for red fluorescent protein. We were able to see a ten fold repression upon increasing the concentration of siRNA from 0 to 1 nM. Saturation appeared at about 10 nM. (graph)
More information on testing target site sensitivity.

Why use a miRNA sensor?

The two most common methods for looking at the miRNA profile of a cell line are by miRNA microarray and quantitative polymerase chain reaction (qPCR). However, it has been indicated that the physical amount of miRNA present doesn't always have a strong correlation to the repression of the associated gene. By using a miRNA sensor, we can directly measure the activity of the miRNA, which the aspect of miRNA being used in the circuit.

"We detected the expression of more than 310 miRNAs (Fig. 2a). Our library included sensors for 165 of these miRNAs (188 when considering families), but we detected the suppression of only 67 sensors (Fig. 2b). Thus, 59% of the expressed miRNAs that we sampled did not have suppressive activity." Mullokandov et. al Nature 2012

Using a miRNA Sensor

The sensor, courtesy of Jeremy Gam, consists of a red fluorescent protein, mKate, which is constitutively controlled by the hef1a promoter. After mKate, you will notice here are four yellow blocks. These represent four miRNA target site domain repeats. After the target sites is a blue fluorescent protein also controlled by the hef1a promoter. When there is miRNA present, it will bind to the miRNA target site on the mKate mRNA and repress the expression of mKate.

Probing tHESC for miRNA Levels

Unlike more common cell lines like MCF7 and HEK293, tHESC is not a highly characterized cell line. This meant that the levels of our eight miRNA candidates were completely unknown in tHESC. We set out to characterize our miRNA target sites (miRNA-ts) in tHESC using a miRNA sensor. What is tHESC?