RNA: Tools for Cell Biology

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

This year we reported the structural basis of function of a novel fluorogenic RNA aptamer called Squash. Thus far, most fluorescence turn-on RNA aptamers have been evolved in vitro starting with random sequence libraries. While this approach is in principle unbiased, it requires that RNA structure and function arise concomitantly in one evolution experiment. This stringent requirement may bias the evolution experiments to RNA folds that are abundant in sequence space, to the detriment, for instance of the ability to function in live cells. Indeed, the majority of fluorogenic RNAs characterized to date employ the G-quadruplex, an unusually stable RNA secondary structure element to assemble their fluorophore binding sites. To examine if an in vitro evolution experiment that starts with a naturally-evolved structured RNA element, rather than random RNA, could produce a fluorescence turn-on RNA with improved properties, the Jaffrey laboratory previously selected Squash, starting with the stably folding bacterial adenine riboswitch aptamer domain. We now determined the crystal structure of Squash bound to its cognate fluorophores, and discovered that while this RNA retains the overall fold of the bacterial RNA, it has extensively rearranged it central ligand-binding area. Moreover, Squash does not employ a G-quadruplex in its fluorophore binding pocket. The mechanism of fluorophore binding by Squash is also remarkable, because unlike other fluorogenic RNAs (and fluorescent proteins, such as GFP, that activate structurally related fluorophores) this RNA has a ligand binding pocket that is largely apolar. Our structural characterization of Squash is the starting point for further, structure-guided optimization efforts, and also sets the stage for the engineering of gene-regulatory RNAs that are selectively activated by fluorophores, and turn on fluorescence when they are activating a gene. Such tools would open new possibilities in the field of synthetic biology.