RNA: Tools for Cell Biology

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Our collaborators in the Unrau laboratory at Simon Fraser University had previously reported the development of the fluorescent RNA module called 'Mango-I', but further improvement was hampered by a complete lack of knowledge of the structural and biophysical basis for function. In 2016-2017, we determined the crystal structure of Mango-I, and discovered that this fluorogenic RNA binds its chromophore between a three-tiered G-quadruplex and three flap nucleotides. Consistent with the modest quantum yield of the RNA-dye complex, the two heterocycles of the latter are not in plane. Since a planar arrangement is predicted to maximize quantum yield this immediately suggests avenues for further improving the fluorescence properties of RNA Mango. In this period, we have determined the crystal structure of one such improved version, Mango-II, which achieves a quantum yield that is three times higher. The basic structural scaffold is the same as that of the parental aptamer, but Mango-II has a different arrangement of the flap nucleotides, making it also a good turn-on aptamer for a red-shifted fluorophore. In complex with this, Mango-II is one of the most red-shifted fluorogenic tags described. In this period we also reported the structure of 'Corn' an RNA selected by our collaborators in the Jaffrey laboratory at Cornell University. Our structure revealed an unexpected homodimeric RNA, which is globally symmetric but is locally asymmetric at the interface, where one molecule of its cognate fluorophore is bound. This asymmetry forms the starting point for the development of obligate heterodimeric variants, which would function analogously to split GFP and may be useful for detecting co-localization and co-expression of non-coding RNAs in live cells.