Isolation and characterization of fluorophore-binding RNA aptamers

Abstract

Background: In vitro selection has been shown previously to be a powerful method for isolating nucleic acids with specific ligand-binding functions (‘aptamers’). Given this capacity, we have sought to isolate RNA motifs that can confer fluorescent labeling to tagged RNA transcripts, potentially allowing in vivo detection and in vitro spectroscopic analysis of RNAs.

Results: Two aptamers that recognize the fluorophore sulforhodamine B were isolated by the in vitro selection process. An unusually large motif of approximately 60 nucleotides is responsible for binding in one RNA (SRB-2). This motif consists of a three-way helical junction with two large, highly conserved unpaired regions. Phosphorothioate mapping with an iodoacetamide-tagged form of the ligand shows that these two regions make close contacts with the fluorophore, suggesting that the two loops combine to form separate halves of a binding pocket. The aptamer binds the fluorophore with high affinity, recognizing both the planar aromatic ring system and a negatively charged sulfonate, a rare example of anion recognition by RNA. An aptamer (FB-1) that specifically binds fluorescein has also been isolated by mutagenesis of a sulforhodamine aptamer followed by re-selection. In a simple in vitro test, SRB-2 and FB-1 have been shown to discriminate between sulforhodamine and fluorescein, specifically localizing each fluorophore to beads tagged with the corresponding aptamer.

Conclusions:In addition to serving as a model system for understanding the basis of RNA folding and function, these experiments demonstrate potential applications for the aptamers in transcript double labeling or fluorescence resonance energy transfer studies.