title: Strategy for Internal Labeling of Large RNAs with Minimal Perturbation by Using Fluorescent PNA.
abstract: Fluorescence techniques for the investigation of biomolecules and their folding pathways require an efficient labeling strategy. A common method to internally label large RNAs involves the introduction of long loops for hybridization of fluorophore-carrying DNA strands. Such loops often disturb the structure, and thus the functionality, of the RNA. Here we show, in a proof of concept study with a >600 nucleotide group II intron ribozyme, that the usage of the nucleic acid analogue peptide nucleic acid (PNA) is more efficient in several aspects, minimizing the required structural modifications of the RNA. We demonstrate by various methods, including smFRET, that much smaller concentrations and shorter PNAs can be applied, compared to DNA, for rapid and specific internal RNA labeling. The folding pathway and catalytic activity of this large ribozyme is only minimally affected by the PNA, but the background signal is significantly reduced.
authors: Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich (Switzerland).
authors: Anita G
authors: AG
authors: Schmitz
authors: Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich (Switzerland).
authors: Susann
authors: S
authors: Zelger-Paulus
authors: Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich (Switzerland). gilles.gasser@chem.uzh.ch.
authors: Gilles
authors: G
authors: Gasser
authors: Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich (Switzerland). roland.sigel@chem.uzh.ch.
authors: Roland K O
authors: RK
authors: Sigel
mesh_terms: D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D016337:RNA, Catalytic / Q000032:analysis*; D012331:RNA, Fungal / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D016337:RNA, Catalytic; D012331:RNA, Fungal; C085321:cyanine dye 5
keywords: FRET; RNA; peptide nucleic acids; ribozymes; single-molecule studies