A homodimer interface without base pairs in an RNA mimic of red fluorescent protein

Abstract

Corn, a 28-nucleotide RNA, induces yellow fluorescence of its cognate ligand (3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, DFHO) by >1000-fold. It was selected in vitro to overcome limitations of other fluorogenic RNAs, particularly rapid photobleaching. We now report the Corn-DFHO co-crystal structure, discovering that the functional species is a quasisymmetric homodimer. Unusually, the dimer interface, where six unpaired adenosines break overall 2-fold symmetry, lacks any intermolecular base pairs. The homodimer encapsulates one DFHO at its inter-protomer interface, sandwiching it with a G-quadruplex from each protomer. Corn and the green-fluorescent Spinach RNA are structurally unrelated. Their convergent use of G-quadruplexes underscores the usefulness of this motif for RNA-induced small-molecule fluorescence. The asymmetric dimer interface of Corn could form the basis for the development of mutants that only fluoresce as heterodimers. Such variants would be analogous to Split GFP, and may be useful in analyzing RNA co-expression or association, or in designing self-assembling RNA nanostructures.

RNA Localization in Bacteria

“…The Spinach system (Figure 1C) is an example of the first generation of RNA aptamers for live-cell imaging (52). Since then, new aptamer systems have been developed, including Spinach 2 (53), Mango (54), Broccoli (55, 56), and Corn (57). Repetitive Spinach aptamers have been shown to increase the brightness by a maximum of 17-fold (with 64 repeats) compared to a single Spinach monomer at a cost of a significant lengthening of the aptamer sequence (58).…”

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

“…Herein, the bimolecular interaction of two molecules of interest brings the two fluorophores into close proximity,s o that the direct transfer of the excited donor electron can excite an electron of the acceptor molecule.T wo observable readouts are commonly used:1)the fluorescence emission of the donor molecule decreases as the acceptor molecule takes over the energy of the excited electron, and 2) the acceptor molecule can also release this energy through fluorescence of au sually red-shifted wavelength. [71] Apta-FRET could be shown in vitro and also in vivo when expressed in bacteria (Figure 10 D). Tw oaptamers with suitable spectral characteristics (Spinach and Mango) were fused together in as ystem called "apta-FRET" (Figure 10 C).…”

“…[71] However,i ts functional unit is ah omodimer,t hus making it similar to the size of other Spinach-based aptamers.I ts 3D structure resembles as tem-loop with as hort but stable stem ( Figure 7A,P 1), al inker region J1, and al oop region that folds back onto itself (tip). [71] However,i ts functional unit is ah omodimer,t hus making it similar to the size of other Spinach-based aptamers.I ts 3D structure resembles as tem-loop with as hort but stable stem ( Figure 7A,P 1), al inker region J1, and al oop region that folds back onto itself (tip).…”

“…[71] However,i ts functional unit is ah omodimer,t hus making it similar to the size of other Spinach-based aptamers.I ts 3D structure resembles as tem-loop with as hort but stable stem ( Figure 7A,P 1), al inker region J1, and al oop region that folds back onto itself (tip). [71] TheD FHO fluorogen is coordinated on top of this G-quadruplex region, thereby locking it into its planar conformation. Thes equence of nucleotides composing the G-quadruplexes is non-consecutive,but in contrast to Spinach G-quadruplexes,t he interlinking loops are small (1 nt), with the biggest being the folding back tip (5 nt, Figure 7B).…”

“…FLAPs are fluorophores that in principle can undergo such mechanism. [71] [71] Thefusion of the two,aswell as several steps of construct optimization (kissing loop structure,v ariations of scaffolding RNA-duplex) for close proximity and correct dipole orientation, fluorophore optimization for better spectral overlap (oxazole yellow derivative YO3-biotin as Mango fluorogen instead of TO1-biotin, Figure 2B), resulted in an RNAc onstruct with two FLAPs that undergo FRET using Mango fluorescence as ar ead-out of the acceptor.…”