A practical approach to FRET-based PNA fluorescence in situ hybridization

Given the demand for improved methods for detecting and characterizing RNA variants in situ, we developed a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction as FRET efficiency measure. The FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location, and dynamics but also potentially a wide variety of close range RNA–RNA interactions. In this paper, we explain the FP-FISH technique workflow for reliable and reproducible results.     

A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization

We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5–11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP–FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA–RNA interactions.     

Preparation of radioiodinated peptide nucleic acids with high specific activity

Peptide nucleic acids (PNAs) have stronger affinity and greater specificity than do oligonucleotides for binding to DNA and RNA and, as such, have potential utility as probes in molecular biology applications. In this study, a novel approach for labeling the PNA with radioiodine that avoided solubility issues and poor labeling encountered when trying to radioiodinate PNAs directly in solution was developed. For this approach, a purpose-designed prosthetic group that incorporated both a radioiodinatable tyrosine and a triphenylphosphonium (TPP) moiety was synthesized. The latter is an organic cation that combines the properties of good solubility in both aqueous and organic solvents with a strong retention by reverse phase HPLC. Following radioiodination of the TPP-based prosthetic group in phosphate buffer, the prosthetic group was purified and coupled to the terminal amine of 15-mer PNA on the solid phase resin. After cleavage and deprotection of the PNA from the resin, the presence of the TPP group resulted in a clean separation of radioiodinated PNA from unlabeled PNA, yielding a high-specific activity probe in a single HPLC run. As an example of a potential molecular biology application of the resultant (125)I-labeled PNA probe, it was used to detect mRNA for the Lcn2 gene in Northern blotting.     

Real time RNA transcription monitoring by Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes: norovirus detection

Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes have been reported as a valuable strategy for DNA analysis; however, no investigations targeting RNA molecules and no comparisons between different derivatization approaches have been reported so far. In this work, two TO-conjugated PNAs for genogroup II noroviruses (NoV GII) detection were designed and synthesized. Both the probes target the most conserved stretch of nucleotides identified in the open reading frame 1-2 (ORF1-ORF2) junction region and differ for the dye conjugation strategy: one PNA is end-labelled with the TO molecule tethered by a linker; the other probe bears the TO molecule directly linked to the PNA backbone, replacing a conventional nucleobase. The spectroscopic properties of the two PNA probes were studied and their applicability to NoVs detection, using an isothermal assay, was investigated. Both probes showed good specificity and high fluorescence enhancement upon hybridization, especially targeting RNA molecules. Moreover, the two probes were successfully employed for NoVs detection from stool specimens in an isothermal-based amplification assay targeting RNA 'amplicons'. The probes showed to be specific even in the presence of high concentrations of non-target RNA.     

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.     

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.     

Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle

Steric blocking peptide nucleic acid (PNA) oligonucleotides have been used increasingly for redirecting RNA splicing particularly in therapeutic applications such as Duchenne muscular dystrophy (DMD). Covalent attachment of a cell-penetrating peptide helps to improve cell delivery of PNA. We have used a HeLa pLuc705 cell splicing redirection assay to develop a series of PNA internalization peptides (Pip) conjugated to an 18-mer PNA705 model oligonucleotide with higher activity compared to a PNA705 conjugate with a leading cell-penetrating peptide being developed for therapeutic use, (R-Ahx-R)₄. We show that Pip–PNA705 conjugates are internalized in HeLa cells by an energy-dependent mechanism and that the predominant pathway of cell uptake of biologically active conjugate seems to be via clathrin-dependent endocytosis. In a mouse model of DMD, serum-stabilized Pip2a or Pip2b peptides conjugated to a 20-mer PNA (PNADMD) targeting the exon 23 mutation in the dystrophin gene showed strong exon-skipping activity in differentiated mdx mouse myotubes in culture in the absence of an added transfection agent at concentrations where naked PNADMD was inactive. Injection of Pip2a-PNADMD or Pip2b-PNADMD into the tibealis anterior muscles of mdx mice resulted in ~3-fold higher numbers of dystrophin-positive fibres compared to naked PNADMD or (R-Ahx-R)₄-PNADMD.