Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness

Abstract

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.

Live cell imaging of single RNA molecules with fluorogenic Mango II arrays

“…We chose the Mango II aptamer (Fig. 1a) because of its resistance to formaldehyde fixation, enhanced thermal stability and high affinity for TO1-Biotin (TO1-B) 28,29 . As Mango II requires a closing stem to efficiently fold, we engineered modified stem sequences by altering the Watson-Crick base pairs of three adjacent aptamers to avoid potential misfolding between the aptamer modules ( Fig.…”

From fluorescent proteins to fluorogenic RNAs: Tools for imaging cellular macromolecules

“…The open pocket also accommodates the TO3 headgroup, explaining the similar affinity of both fluorophores, 1.1 and 1.3 n M ). Biotin is not present in the ligand binding pocket, and while most of the PEG linker appeared to be excluded from the pocket, it was shown that its interactions with the RNA contribute to affinity and selectivity …”

Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes

“…47,57,58 The structures of Mango variants from generations I-IV are known, and have recently been discussed in detail. 3,[58][59][60][61] The core of all Mango aptamers contains a G-quadruplex, primarily responsible for stacking and rigidification of the bound ligand. While the first generation included the PEG linker and the biotin in packing onto the G-quartet next to TO1, 61 later generations exhibited improved fluorophore planarity resulting in higher brightness.…”