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Strategy for Internal Labeling of Large RNAs with Minimal Perturbation by Using Fluorescent PNA
Author(s) -
Schmitz Anita G.,
ZelgerPaulus Susann,
Gasser Gilles,
Sigel Roland K. O.
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500180
Subject(s) - ribozyme , rna , nucleic acid , peptide nucleic acid , fluorophore , dna , biomolecule , deoxyribozyme , nucleotide , biochemistry , nucleic acid structure , chemistry , fluorescence , g quadruplex , folding (dsp implementation) , biophysics , biology , computational biology , gene , physics , quantum mechanics , electrical engineering , engineering
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.