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May 10, 2012

Photoreactive Probe Technique Identifies mRNA Targets of miRNAs in Live Cells

  • Scientists have devised a simple technique for identifying the mRNA targets of specific miRNAs directly in normal or diseased live cells. The approach, called miRNA target RNA affinity purification (miR-TRAP), doesn’t use antibodies, so minimizes nonspecific background signals. And in addition to identifying mRNA targets that are downregulated by a specific miRNA , it can also identify those mRNAs whose translation is repressed by an miRNA and that can’t be identified by existing techniques such as quantitative PCR and microarray analysis.

    miR-TRAP has been developed by a team at Sanford-Burnham Medical Research Institute. The approach basically turns miRNA molecules into photoreactive probes by conjugating them to psoralen (Pso). The miRNA-psoralen probes interact with their target mRNAs in a cell, and the cells are then exposed to UVA radiation, which causes the PSo molecule on the miRNA mimic to react with the uridine on its target mRNA, generating a covalently bound complex. The miRNA-mRNA complex can then be recovered using biotin-streptavidin affinity purification and the mRNA target identified.

    Sandford-Burnham’s Tariq M. Rana, Ph.D., and colleagues claim a major advantage of the miR-TRAP technique is that photocrosslinking using Pso requires a UV radiation wavelength of 360 nm, which is less damaging to cells than the shorter, 254 nm UV radiation used in another miRNA target identification technique known as HITS-CLIP. The Pso-crosslinked product can also be photoreversed once isolated, by treatment with short wave UV radiation.

    The researchers describe their technique in Angewandte Chemie International Edition in a paper titled “miR-TRAP: A Benchtop Chemical Biology Strategy to Identify microRNA Targets.” The team used the platform to analyze 13 predicted targets of two important miRNAs: miRNA-135b and miRNA-29a. As well as identifying the known mRNA targets for both the miRNAs, miR-TRAP also identified two new targets; Elk3 for miRNA-135b and Tet2 for miRNA-29a.

    The researchers hope that miR-TRAP will aid in the discovery of miRNA targets in a range of diseases, and are applying the technique to a number of disease models. “miR-TRAP will help bridge a gap in the RNA field, allowing researchers to better understand diseases like cancer and target their genetic underpinnings to develop new diagnostics and therapeutics,” Dr. Rana states. “This will become especially important as new high-throughput RNA sequencing technologies increase the numbers of known miRNAs and their targets.” 


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