Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity

<div><p>A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and <i>vice versa</i>) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.</p></div

Hybridization of anti-parallel complementary oligonucleotides is stereospecific.

<p>Schematic representation of (A) hammerhead ribozyme and (B) DNAzyme in complex with the target RNA sequence (green). Hydrolysis sites are indicated by arrows. To analyze (potential) hybridization of enantiomeric, anti-parallel complementary oligonucleotides (3 μM each in 10 mM phosphate buffer, pH 7.4, 100 mM NaCl) temperature-dependent hyperchromicity at 260 nm was measured. (C) d-RNA and (D) l-RNA with anti-parallel complementary d-RNA (red) and l-RNA (blue). (E) d-RNA and (F) l-RNA with anti-parallel complementary d-DNA (red) and l-DNA (blue). (G) d-DNA and (H) l-DNA with anti-parallel complementary d-DNA (red) and l-DNA (blue). Mean of three melting ramps (25°C to 95°C) is given as normalized absorption A / Amax at 260 nm. First derivative is shown as dotted line. Data are representative of three independent experiments.</p

Stereospecificity of ribozyme and DNAzyme activity.

<p>5′-fluorescein-labeled d- or l-RNA substrate (200 nM) was incubated with (A) d- or l-hammerhead ribozyme (2 μM) or (B) d- or l-DNAzyme (2 μM) in 50 mM Tris (pH 7.5), 10 mM MgCl₂ for 5 h at 37°C. RNA substrate and its 5′-hydrolysis products were visualized via a 5′-fluorescein tag. Ribozymes and DNAzymes were stained with ethidium bromide. Data is representative of three independent experiments.</p

Ribozyme cleavage is prone to enantiomeric contaminations.

<p>l-hammerhead ribozyme solution (2 μM) was artificially contaminated with enantiomeric d-ribozyme (20 nM–200 fM) and cleavage of d-RNA substrate was analyzed after 5 h at 37°C. Assays were performed in 50 mM Tris, pH 7.5 in the presence of (A) 10 mM MgCl₂ and (B) 1 mM MgCl₂. RNA substrate and its 5′-hydrolysis products were visualized via a 5′-fluorescein tag. Ribozymes and DNAzymes were stained with ethidium bromide. Data is representative of two independent experiments.</p

PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells

Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 °C) as well as real-time PCR measurements (at 60 °C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species

Liquid Crystal Ordering and Isotropic Gelation in Solutions of Four-Base-Long DNA Oligomers

Liquid crystal ordering is reported in aqueous solutions of the oligomer 5′-ATTAp-3′ and of the oligomer 5′-GCCGp-3′. In both systems, we quantitatively interpret ordering as stemming from the chaining of molecules <i>via</i> a “running-bond” type of pairing, a self-assembly process distinct from the duplex aggregation previously reported for longer oligonucleotides. While concentrated solutions of 5′-ATTAp-3′ show only a columnar liquid crystal phase, solutions of 5′-GCCGp-3′ display a rich phase diagram, featuring a chiral nematic phase analogous to those observed in solutions of longer oligonucleotides and two unconventional phases, a columnar crystal and, at high concentration, an isotropic amorphous gel. The appearance of these phases, which can be interpreted on the basis of features of 5′-GCCGp-3′molecular structure, suggests distinctive assembly motifs specific to ultrashort oligonucleotides