miRNA Target Validation

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Bioinformatics identification of miRNA targets is challenging. Most of the algorithms use comparative genomic analysis, searching conserved blocks in 3'UTRs for the presence of potential miRNA seeds, a 7nt sequence complementary required for miRNA site recognition, and then extend the seed testing to other features, such as the thermodynamic stability of the paired sequences. However, miRNAs can pair with targets that are slightly different each time, suggesting that the perfect seed pairing is not always a good predictor of the presence of a target. Characterizing the network of genes that miRNAs such as let-7 target would advance not only our understanding of the intricacies of miRNA biology, but more importantly, could help identify the contribution of miRNAs in disease, providing a foundation for developing targeted therapeutics. The major obstacle in establishing these kinds of networks is the lack of high-throughput tools that can be used to screen multiple putative miRNAs in a single experiment and detect their targets in parallel. GFP reporters and other assays are the standard molecular biology approaches used to detect and validate these functional elements, but the techniques are specifically designed to detect a single pair of interaction each time and are difficult to scale up maintaining affordable costs.

3’LIFE Assay

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We are developed a new technology that will allow the rapid and systematic screen of functional miRNA targets in human 3'UTRs using a high-throughput approach.
The system, that we have named 3'-LIFE (Luminescence-based Identification of Functional Elements in 3'UTRs), overcomes the limitations of scalability and resolution of current methods, allowing the systematic and inexpensive mapping of regulatory elements in 3'UTRs.
3'-LIFE is an adaptation of the well characterized dual luciferase reporter assay, but is scaled up and performed in high-throughput using 96-well plates handled by dedicated robots, together with robust bioinformatic analysis and wet-bench approaches. A major advantage of 3'LIFE is that it is a functional assay, meaning that the elements are detected and validated at the same time. The 3'LIFE assay is based on the dual-luciferase assay, a sensitive, rapid, and easily scalable assay to detect fluctuations in protein expression.
The dual luciferase assay relies on the fusion of a test 3'UTR to a luminescent reporter gene. Targeting and translational repression of the test 3'UTR by a probe miRNA is identified by a decrease in the luciferase::3'UTR signal (Firefly luciferase) relative to a second normalization signal (Renilla luciferase). 3'LIFE is designed to rapidly identify functional targets of a given miRNA in a panel of hundreds of test 3'UTRs in co-transfection experiments. The luminescence of each luciferase protein is measured sequentially in each well, and used to quantify translation of the reporter::3'UTR. Putative miRNA targets (red spots) are assigned when protein levels change in response to cotransfection with a miRNA compared to a negative control (blue spots).