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.

Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy

“…1. Fluorescently labelled (and biotinylated) DNA oligonucleotides were purchased from IBA (Göttingen, Germany), and PNA oligonucleotides were synthesized as described elsewhere [12]. Any molecule carrying a dye, always needs to be protected from light.…”

“…3. Add fluorescently labelled DNA [11] or PNA oligonucleotides [12] carrying each a sulfonated fluorophore, the donor (sCy3) and acceptor (sCy5), which are complementary to the respective labelling sites, in a 1:1 ratio (see Note 5) to the RNA for hybridization illustrated in Figure 2 (see Note 6).…”

“…The immobilization to a functionalized and passivated surface via biotin-streptavidin linkage implies RNA modifications to hybridize a biotin-carrying DNA or PNA oligonucleotide [11,12] or to covalently link a biotin-moiety to the RNA [13]. Molecule immobilization can cause interactions between the molecule and the surface, which might influence the molecule's fold, and thus leads to misfolding or changes in the associated activity [14].…”

“…In contrast to DNA, PNA are without charge and thus have a higher binding affinity towards RNA. For that reason, PNAs can be kept much shorter (down to 8 -10 nt) and retain similar hybrid stability [12].…”

Fluorescent peptide displacement as a general assay for screening small molecule libraries against RNA

“…Conventional fluorescence assays, however, frequently involve incorporation of fluorescent probes in the RNA sequence, which can alter the native structure and often present synthetic challenges. [17][18][19] One prominent screening method against nucleic acids is the fluorescent indicator displacement assay (FID) where the indicator displays different fluorescence properties in the presence and absence of the oligonucleotide and thus can be utilized to measure the binding properties of small molecules. 20 Common fluorescent probes used in FID assays for nucleic acids include intercalating dyes such as ethidium bromide, Thiazole Orange (TO), and TO-PRO-1.…”