Co-crystal structures of the fluorogenic aptamer Beetroot show that close homology may not predict similar RNA architecture

Luiz F. M. Passalacqua, Mary R. Starich, Katie A. Link, Jiahui Wu, Jay R. Knutson, Nico Tjandra, Samie R. Jaffrey and Adrian R. Ferré-D’Amaré ()
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Luiz F. M. Passalacqua: National Institutes of Health
Mary R. Starich: National Institutes of Health
Katie A. Link: National Institutes of Health
Jiahui Wu: Cornell University
Jay R. Knutson: National Institutes of Health
Nico Tjandra: National Institutes of Health
Samie R. Jaffrey: Cornell University
Adrian R. Ferré-D’Amaré: National Institutes of Health

Nature Communications, 2023, vol. 14, issue 1, 1-12

Abstract: Abstract Beetroot is a homodimeric in vitro selected RNA that binds and activates DFAME, a conditional fluorophore derived from GFP. It is 70% sequence-identical to the previously characterized homodimeric aptamer Corn, which binds one molecule of its cognate fluorophore DFHO at its interprotomer interface. We have now determined the Beetroot-DFAME co-crystal structure at 1.95 Å resolution, discovering that this RNA homodimer binds two molecules of the fluorophore, at sites separated by ~30 Å. In addition to this overall architectural difference, the local structures of the non-canonical, complex quadruplex cores of Beetroot and Corn are distinctly different, underscoring how subtle RNA sequence differences can give rise to unexpected structural divergence. Through structure-guided engineering, we generated a variant that has a 12-fold fluorescence activation selectivity switch toward DFHO. Beetroot and this variant form heterodimers and constitute the starting point for engineered tags whose through-space inter-fluorophore interaction could be used to monitor RNA dimerization.

Date: 2023
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DOI: 10.1038/s41467-023-38683-3

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