2 research outputs found
Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness
Several RNA aptamers
that bind small molecules and enhance their
fluorescence have been successfully used to tag and track RNAs <i>in vivo</i>, but these genetically encodable tags have not yet
achieved single-fluorophore resolution. Recently, Mango-II, an RNA
that binds TO1-Biotin with ā¼1 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-IIĀ(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 fluorophoreāRNA interface. Our
work demonstrates that even after extensive functional selection,
aptamer RNAs can be further improved through structure-guided engineering
RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking
Because RNA lacks strong intrinsic
fluorescence, it has proven
challenging to track RNA molecules in real time. To address this problem
and to allow the purification of fluorescently tagged RNA complexes,
we have selected a high affinity RNA aptamer called RNA Mango. This
aptamer binds a series of thiazole orange (fluorophore) derivatives
with nanomolar affinity, while increasing fluorophore fluorescence
by up to 1,100-fold. Visualization of RNA Mango by single-molecule
fluorescence microscopy, together with injection and imaging of RNA
Mango/fluorophore complex in <i>C. elegans</i> gonads demonstrates
the potential for live-cell RNA imaging with this system. By inserting
RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating
the fluorophore, we demonstrate that the aptamer can be used to simultaneously
fluorescently label and purify biologically important RNAs. The high
affinity and fluorescent properties of RNA Mango are therefore expected
to simplify the study of RNA complexes