Fluorogenic aptamers resolve the flexibility of RNA junctions using orientation-dependent FRET

Abstract

To further understand the transcriptome, new tools capable of measuring folding, interactions, and localization of RNA are needed. Although Förster resonance energy transfer (FRET) is an angle- and distance-dependent phenomenon, the majority of FRET measurements have been used to report distances, by assuming rotationally averaged donor–acceptor pairs. Angle-dependent FRET measurements have proven challenging for nucleic acids due to the difficulties in incorporating fluorophores rigidly into local substructures in a biocompatible manner. Fluorescence turn-on RNA aptamers are genetically encodable tags that appear to rigidly confine their cognate fluorophores, and thus have the potential to report angular-resolved FRET. Here, we use the fluorescent aptamers Broccoli and Mango-III as donor and acceptor, respectively, to measure the angular dependence of FRET. Joining the two fluorescent aptamers by a helix of variable length allowed systematic rotation of the acceptor fluorophore relative to the donor. FRET oscillated in a sinusoidal manner as a function of helix length, consistent with simulated data generated from models of oriented fluorophores separated by an inflexible helix. Analysis of the orientation dependence of FRET allowed us to demonstrate structural rigidification of the NiCo riboswitch upon transition metal-ion binding. This application of fluorescence turn-on aptamers opens the way to improved structural interpretation of ensemble and single-molecule FRET measurements of RNA.

RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

“…1 D,E, Supplementary Table 2). This decrease can either result from a distance effect or an oriented dipole effect as has been documented previously 6,26 . When placing the aptamers further apart on helix 1 and 3 in the 1,3-B12P12 design, we observe a FRET output of ∼10% and when placing the aptamers outside FRET distance in the 1,3-B12P(−34) design, no FRET was observed (Fig.…”

“…Förster resonance energy transfer (FRET) represents a versatile tool to characterize scaffolding effects and build devices sensitive to small structural changes, having applications in studies of localization of molecules and conformational changes [23][24][25][26] . Fluorogenic aptamers (FAs) are RNA sequences that interact with and activate fluorophores, reducing their nonradiative decay through vibrational and rotational relaxation by rigidifying their aromatic orbital systems 27,28 .…”

“…Apta-FRET has also been demonstrated with the iSpinach and Mango-IV aptamers joined together by a single-stranded region 30 . Jeng et al observed angular changes in an apta-FRET system and were able to sense the structural stabilization of a metal-ion binding riboswitch 26 . Geary et al used RNA origami to demonstrate controlled positioning of Spinach and Mango aptamers and effects of helix length and flexibility on FRET efficiency 14 .…”

RNA origami design tools enable cotranscriptional folding of kilobase-sized nanoscaffolds

“…5i ) (statistically significant p<0.05 in Student’s t-test except for L2T-3DT). Within this series, care was taken to maintain the relative orientation of the Spinach and Mango aptamers to avoid the possible effects of oriented dipoles on FRET 55 ( Supplementary Fig. 29 ).…”

RNA Peach and Mango: orthogonal two-color fluorogenic aptamers distinguish nearly identical ligands

“…Using pairs of fluorogenic aptamers is in its infancy; RNA Mango and RNA Spinach ( Paige et al 2011 ) or its close relatives ( Filonov et al 2014 ; Song et al 2017 ) have been used in the development of apta-FRET two channel imaging ( Jepsen et al 2018 ; Jeng et al 2020 ). The Spinach family of aptamers, with its green ligand 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), have relatively weak fluorophore binding affinity and low ligand discrimination ( Jeng et al 2016 ), making the optimization of imaging conditions challenging.…”