miRNA Target Validation
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 impo
rtantly, 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.
We are developing 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.