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Broccoli sheds light on RNA-modifying enzymes

Friday, April 1, 2016

Dr Samie JaffreyDr. Samie Jaffrey Photo credit: Robert Essel RNA-modifying enzymes affect a variety of cellular processes. For example, the fat mass and obesity associated protein (FTO)is a demethylase that targets N6-methyladenosine (m6A), and variations in the FTO gene can cause brain and developmental malformations linked to obesity. Despite their potential as therapeutic targets, RNA-modifying enzymes remain difficult to study.

A fluorometric assay would aid high-throughput screens for drug development, but none are available yet because it is difficult to couple RNA base modifications with changes in fluorescence. Now, writing in in Cell Chemical BiologyNina Svensen and Samie Jaffrey at Weill Cornell Medical College describe Broccoli, a derivative of the Spinach fluorescent aptamer for assaying RNA-modifying enzymes.

“There is definitely a value for drug screening. A system is needed that can evaluate enzymes that add or remove base modifications.” Jaffrey said. “There are systems right now, but they are incredibly slow. Our assay can be adapted to other nucleotides and can benefit drug development as well as basic scientists.”

Svensen and Jaffrey modified Spinach by introducing m6A modifications that FTO can recognize and demethylate. Methylated Broccoli cannot bind its cognate fluorophore, so it fluoresces only after FTO demethylates it.

Using Broccoli, the researchers performed a high-throughput screen of a library of 1280 diverse small organic compounds and identified 12 novel FTO inhibitors. They were able to validate the specificity of several of these inhibitors in vivo.

For researchers who would like to adapt the system for other enzymes, Jaffrey recommends benchmarking aptamers with HPLC and working in RNase-free conditions. He also mentioned that specificity may be a limitation for working with other enzymes. “If the sequence context of the modified nucleotides is not tolerated by Broccoli, then it won’t work, but it should be straightforward for most labs,” he said.

The team now plans to conduct a much larger screen, and they are interested in designing aptamers for expression in living cells to look for fluorescence upon nucleotide modification or m6A demethylation. "This would reveal spatial and temporal dynamics of RNA modification in living cells,” he said.

This article first appeared in BioTechniques.