RNA: Tools for Cell Biology

National Heart, Lung, And Blood Institute

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Abstract

In the past year, we reported the structure-based design and biophysical, biochemical and structural characterization of ultrabright-next-generation fluorophores for RNA imaging in vitro and in vivo. Previously, we have extensively characterized fluorogenic aptamer RNAs. These in vitro evolved molecules bind and selectively turn on the fluorescence of otherwise non-fluorescent small molecule ligands. Some fluorogenic aptamers recognize their cognate ligands with low-nanomolar dissociation constants and fluorescence turn-on ratios (bound vs. unbound fluorophore) of over 5000. Thus, they are powerful tools for imaging RNAs in vitro and in vivo, as RNA analogs of green fluorescent protein. In order to obtain ultra-bright fluorophores, we carried out structure-guided design combined with medicinal chemistry of fluorophores targeted to the fluorogenic RNAs of the "Mango" family. A variety of fluorophores were synthesized. obtained. One, in particular, despite being highly similar to the parental fluorophore, exhibited fluorescence much higher. Crystallographic structure determination of this, currently the brightest known fluorophore when activated by a Mango family RNA, revealed how the RNA and its bound cations together lead to the novel properties of the ligand. This work indicates that even small atomic-level changes to RNA-ligand interfaces can have dramatic effects of fluorescence properties, opening new vistas in the development, not only of fluorogenic RNAs, abut RNA-targeted ligands (including drugs) in general.