Andrea Knoll

Fluorescence imaging of influenza H1N1 mRNA in living infected cells using single-chromophore FIT-PNA.

In light of the increasing importance assigned to RNA, significant efforts have been devoted to the development of fluorescent oligonucleotide probes that allow the imaging of RNA expression in living cells. Molecular beacons (MBs, Scheme 1a) probably are amongst the most widely used probes for RNA imaging. These hairpin-shaped probes rely on the interaction between two terminally appended chromophores which are separated upon formation of probe– target complexes. Unintended protein binding and/or nuclease-mediated probe degradation can also affect the distance between the chromophores. We have introduced so-called FIT-PNA probes (Scheme 1 b), which contain a single thiazole orange (TO) intercalator serving as artificial fluorescent nucleobase. These probes respond to changes of the local structure in the vicinity of the dye rather than to the more global changes of conformation that confer fluorescence signaling by the dual-labeled molecular beacons. High fluorescence enhancements require intercalation of the TO dye. This is expected to help in avoiding strong fluorescence signals upon inevitable binding to proteins. Amongst the many TOcontaining probes reported FIT probes are unique because a single fluorophore provides for both high enhancements of fluorescence upon matched hybridization and high target specificity at nonstringent hybridization conditions where both matched and mismatched probe–target complexes coexist. Further assets are the high affinity of the PNA probes for complementary RNA and the enhanced biostability provided by the peptide nucleic acid (PNA) backbone. Herein we demonstrate the advantageous properties of FITPNA probes in the imaging of mRNA from an influenza virus strain belonging to the same subtype as the recently emerged swine virus (A/Mexico/1/2009, H1N1). In a research program aiming at the characterization of the spatio-temporal pattern of virus assembly, we required a method that enables imaging of the mRNA coding for neuraminidase of influenza virus A/PR/8. FIT probes such as 1a and 1b were designed to target a sequence in the NA mRNA (nt 599–615, referred to the accession number NC_002018) which is essentially sequence identical to the NA mRNA from A/Mexico/1/2009/swine (H1N1, nt 625–640) (Figure 1). The accessibility of the target segment, which can be hindered by RNA folding and binding of proteins, has been previously demonstrated by Zhang and co-workers. A rapid screen, which required the synthesis of eight different PNA oligomers, suggested FIT probe 1 a as a suitable probe (see the Supporting Information). This sensor provided an 11-fold increase of the TO emission upon hybridization with complementary RNA target 3a at 37 8C (Figure 2a). Furthermore, we tested the subtype specificity. The RNA 4 from a different influenza strain (NA mRNA from A/X-31, H3N2, nt 16–32) includes seven continuous matched base pairs around the thiazole orange “base”. Nevertheless, the fluorescence of 1a remained virtually unchanged when RNA 4 was added. The TMR/Dabcyl-labeled molecular beacon 2 (Figure 1; TAMRA = tetramethyl-6-carboxyrhodamine; Dabcyl = 4-(4dimethylaminophenyl)diazenylbenzoic acid) was used in a Scheme 1. Nucleic acid detection with a) molecular beacon probes (MBs) and b) FIT-PNA probes. MBs change conformation upon binding of a complementary target. In FIT-PNA probes, an intercalator dye such as thiazole orange (TO) responds to changes of the local environment. Stacking interactions hinder twisting around the TO methine bridge and thus confer enhancements of fluorescence.

Low-Noise Stemless PNA Beacons for Sensitive DNA and RNA Detection†

Fluorescent probes that signal the presence of specific nucleic acids are required in a variety of bioassays, including DNA quantification, SNP typing (SNP = single-nucleotide polymorphism), and analysis of mRNA expression in living cells. The majority of probes take advantage of the distancedependent interaction between two chromophores. Sensitive fluorescent hybridization probes show large hybridizationinduced enhancements of fluorescence emission, which may reach signal-to-background ratios (SBR) on the order of 10. Selective probes enable single-nucleotide-specific fluorescence signaling. Success in both sensitive and specific DNA and RNA detection has been achieved using DNA molecular beacons (MBs, Scheme 1A). These hairpin-shaped probes have been designed to bring the two interacting dyes into close proximity. The SBR is high, because in the absence of target the fluorescence is efficiently quenched by fluorescence resonance energy transfer (FRET), collisional quenching, and/or formation of groundor excited-state complexes. Molecular beacons bind target DNA with high match/ mismatch specificity, but only within a certain temperature range that depends on the difference between thermal stabilities of matched and mismatched probe–target complexes. It is, thus, impossible to distinguish matched from mismatched targets at conditions for which both matched and mismatched probe–target complexes co-exist. The major limitation in molecular-beacon design is that features that increase sensitivity are detrimental to the sequence specificity of fluorescence signaling and vice versa. Large fluorescence enhancements can only be obtained when the stem region is readily opened, while high specificity calls for stable stems that resist opening by mismatched hybridization. We envisioned an alternative beacon design. The approach on one hand retains a signaling mechanism used in molecular beacons, wherein two chromophores detect changes of probe conformation, but it omits the requirement for the formation of stable hairpin structures. On the other hand, “smart” labels are used that become fluorescent and initiate FRET to a near-infrared dye only when the donor dye is embedded in perfectly matched base pairs. It is shown that the combination of the two processes, detection of conformational changes by a switch in energy transfer mechanisms and signaling of altered stacking interactions of an intercalator dye, allows for up to 108-fold fluorescence intensification upon hybridization. Importantly, the stemless probes distinguish matched from mismatched targets at virtually any temperature. Homogeneous detection of both DNA and RNA targets is demonstrated. The design approach is illustrated in Scheme 1B. An intercalator dye, such as thiazole orange, is introduced as base surrogate in a peptide nucleic acid (PNA)-based probe and used as donor for FRET. A terminally appended nearinfrared (NIR) dye, such as NIR667, serves as acceptor dye. It was expected that excitation of the donor in single-stranded probes would induce negligible emission of the acceptor dye because 1) the donor excited state is rapidly depleted owing to torsional motion around the central methine bridge of unstacked thiazole orange, 2) the NIR667 (acceptor) dye is quenched upon collisions with nucleobases, and 3) intramolecular dye–dye dimers or short-lived collision complexes may form, aided by the tendency of the uncharged, hydrophobic PNA molecule to adopt a collapsed structure in Scheme 1. Comparison of A) molecular beacons with B) stemless FIT– PNA beacons in the detection of complementary nucleic acids. In stemless FIT–PNA beacons, an intercalator dye such as thiazole orange (TO) serves as a base surrogate that signals stacking against matched base pairs by FRET to a near infrared dye such as NIR667.

FIT probes: peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery.

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.

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.

Cyclohexyletherδ-Amino Acids: New Leads for Selectivity Filters in Ion Channels

Synthetic ion channels containing δ-amino acids can become cation selective! δ-Amino acids with a cyclohexylether unit were combined with structural motives from gramicidin A and led to channels with a NH4+ /K+ permeability ratio of >10/1. (The current trace for an NH4+ channel is shown.).

Synthesis and functional studies of THF–gramicidin hybrid ion channels

THF-gramicidin hybrids 2-4 with the L-THF amino acid 1 in positions 11 and 12 and compounds 5-8 with the D-THF amino acid ent-1 in positions 10 and 11 were synthesized and their ion channel properties were studied by single-channel-current analysis. The replacement of positions 11 and 12 by the L-THF amino acid 1 gave a strongly reduced channel performance. In contrast, replacement of positions 10 and 11 by the D-THF amino acid ent-1 gave rise to new and interesting channel properties. For the permeability ratios, the ion selectivity shifts from Eisenman I towards Eisenman III selectivity and the channels display ms-dynamics. Most remarkable is the asymmetric compound 8, which inserts selectively into a DPhPC membrane and displays voltage-directed gating dynamics.

Remote Control of Lipophilic Nucleic Acids Domain Partitioning by DNA Hybridization and Enzymatic Cleavage

Lateral partitioning of lipid-modified molecules between liquid-disordered (ld) and liquid-ordered (lo) domains depends on the type of lipid modification, presence of a spacer, membrane composition, and temperature. Here, we show that the lo domain partitioning of the palmitoylated peptide nucleic acid (PNA) can be influenced by formation of a four-component complex with the ld domain partitioning tocopherol-modified DNA: the PNA-DNA complex partitioned into the ld domains. Enzymatic cleavage of the DNA linker led to the disruption of the complex and restored the initial distribution of the lipophilic nucleic acids into the respective domains. This modular system offers strategies for dynamic functionalization of biomimetic surfaces, for example, in nanostructuring and regulation of enzyme catalysis, and it provides a tool to study the molecular basis of controlled reorganization of lipid-modified proteins in membranes, for example, during signal transduction.

Electrophysiological Response of Cultured Trabecular Meshwork Cells to Synthetic Ion Channels

The response of living cells of the trabecular meshwork to synthetic ion channels is described. The THF-gramicidin hybrids THF-gram and THF-gram-TBDPS as well as a linked gA-TBDPS and gramicidin A were applied to cultured ocular trabecular meshwork cells. THF-gram application (minimal concentration, 10(-8) M; saturation, 10(-7) M) led to an additional conductance which displayed characteristics of weak Eisenman-I-selective cation channels, no cell destruction, an asymmetric change of the inward/outward currents, and higher current densities using Cs(+) as charge carrier compared to Na(+) and K(+). Linked-gA-TBDPS showed at 10(-12) M increases of the membrane conductance comparable to gA at 10(-7) M and a much faster response of the cells. Thus, THF-gramicidin hybrids form a basis for the use of synthetic ion channels in biological systems, which eventually may lead to new therapeutic approaches.

Comparing Agent Based Delivery of DNA and PNA FIT-Probes for Multicolor mRNA Imaging

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell‐penetrating peptides (CPPs) and pore‐forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)‐ with DNA‐based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP–FIT PNA conjugates were affected. Live‐cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob‐like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin‐O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA‐based FIT probes were brighter and more responsive than PNA‐based FIT probes. Optimized conditions enabled live‐cell multicolor imaging of three different mRNA target sequences.

Sequence-specific imaging of influenza A mRNA in living infected cells using fluorescent FIT-PNA.

Significant efforts have been devoted to the development of techniques allowing the investigation of viral mRNA progression during the replication cycle. We herein describe the use of sequence-specific FIT-PNA (Forced Intercalation Peptide Nucleic Acids) probes which contain a single intercalator serving as an artificial fluorescent nucleobase. FIT-PNA probes are not degraded by enzymes, neither by nucleases nor by proteases, and provide for both high sensitivity and high target specificity under physiological conditions inside the infected living host cell.