Designed thiazole orange nucleotides for the synthesis of single labelled oligonucleotides that fluoresce upon matched hybridization

Probe molecules that enable the detection of specific DNA sequences are used in diagnostic and basic research. Most methods rely on the specificity of hybridization reactions, which complicates the detection of single base mutations at low temperature. Significant efforts have been devoted to the development of oligonucleotides that allow discrimination of single base mutations at temperatures where both the match and the mismatch probe-target complexes coexist. Oligonucleotides that contain environmentally sensitive fluorescence dyes such as thiazole orange (TO) provide single nucleotide specific fluorescence. However, most previously reported dye-DNA conjugates showed only little if any difference between the fluorescence of the single and the double stranded state. Here, we introduce a TO-containing acyclic nucleotide, which is coupled during automated oligonucleotide synthesis and provides for the desired fluorescence-up properties. The study reveals the conjugation mode as the most important issue. We show a design that leads to low fluorescence of the unbound probe (background) yet permits TO to adopt fluorescent binding modes after the probe-target complex has formed. In these probes, TO replaces a canonical nucleobase. Of note, the fluorescence of the ``TO-base'' remains low when a base mismatch is positioned in immediate vicinity.</p

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

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

Achiral PNA hybridizes to both d- and l-RNA/DNA.

<p>Hybridization of achiral PNA and anti-parallel complementary (A) d-RNA, (B) l-RNA, (C) d-DNA, and (D) l-DNA (3 μM each in 10 mM phosphate buffer, pH 7.4, 100 mM NaCl) was analyzed by measuring temperature-dependent hyperchromicity at 260 nm. Mean of three melting ramps (25°C to 95°C) is given as normalized absorption A / A_max at 260 nm. First derivative is shown as dotted line. Data are representative of three independent experiments.</p

Antisense probes for the experimental control of stereospecificity.

<p>l-ribozyme solution (2 μM) was artificially contaminated with 0.01% enantiomeric d-ribozyme (0.2 nM) and cleavage of d-RNA substrate was analyzed after 5 h at 37°C. d- and l-antisense probes (2 nM) were added to control stereospecificity. Assays were performed in 50 mM Tris, pH 7.5, 10 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

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

Novel Tumor Pretargeting System Based on Complementary l‑Configured Oligonucleotides

Unnatural mirror image l-configured oligonucleotides (L-ONs) are a convenient substance class for the application as complementary in vivo recognition system between a tumor specific antibody and a smaller radiolabeled effector molecule in pretargeting approaches. The high hybridization velocity and defined melting conditions are excellent preconditions of the L-ON based methodology. Their high metabolic stability and negligible unspecific binding to endogenous targets are superior characteristics in comparison to their d-configured analogs. In this study, a radiopharmacological evaluation of a new l-ONs based pretargeting system using the epidermal growth factor receptor (EGFR) specific antibody cetuximab (C225) as target-seeking component is presented. An optimized PEGylated 17mer-L-DNA was conjugated with p-SCN-Bn-NOTA (NOTA′) to permit radiolabeling with the radionuclide 64Cu. C225 was modified with the complementary 17mer-L-DNA (c-L-DNA) strand as well as with NOTA′ for radiolabeling and use for positron emission tomography (PET). Two C225 conjugates were coupled with 1.5 and 5.0 c-L-DNA molecules, respectively. In vitro characterization was done with respect to hybridization studies, competition and saturation binding assays in EGFR expressing squamous cell carcinoma cell lines A431 and FaDu. The modified C225 derivatives exhibited high binding affinities in the low nanomolar range to the EGFR. PET and biodistribution experiments on FaDu tumor bearing mice with directly 64Cu-labeled NOTA′3-C225-(c-L-DNA)1.5 conjugate revealed that a pretargeting interval of 24 h might be a good compromise between tumor accumulation, internalization, blood background, and liver uptake of the antibody. Despite internalization of the antibody in vivo pretargeting experiments showed an adequate hybridization of 64Cu-radiolabeled NOTA′-L-DNA to the tumor located antibody and a good tumor-to-muscle ratio of about 11 resulting in a clearly visible image of the tumor after 24 h up to 72 h. Furthermore, low accumulation of radioactivity in organs responsible for metabolism and excretion was determined. The presented results indicate a high potential of complementary L-ONs for the pretargeting approach which can also be applied to therapeutic radionuclides such as 177Lu, 90Y, 186Re, or 188Re