Lucas Bethge

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.

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.

Single Labeled DNA FIT Probes for Avoiding False-Positive Signaling in the Detection of DNA/RNA in qPCR or Cell Media

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real‐time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance‐dependent interaction between a fluorophore and another label. Such duallabeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label–label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, “DNA FIT probes”, that are designed to avoid false‐positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the “TO base” in cis with the disordered nucleobases of the single strand, and 2) intercalation of the “TO nucleotide” with double strands in trans. However, formation of the probe–target duplex enforces stacking and increases the fluorescence of the TO base. We explored open‐chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe–target complexes. DNA and RNA targets provided up to 12‐fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false‐positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.