7‐Azidomethoxy‐Coumarins as Profluorophores for Templated Nucleic Acid Detection

Templated nucleic acid detection is an emerging bioanalytical method that makes use of the target DNA or RNA strand to initiate a fluorogenic reaction. The Staudinger reduction holds particular promise for templated sensing of nucleic acids because the involved functional groups are highly chemoselective. Here, the azidomethoxy group, which can be removed under Staudinger conditions, is used to cage 7‐hydroxycoumarin fluorophores. Reduction by phosphines and subsequent loss of the azidomethoxy substituent induce a significant bathochromic shift of the major absorbance band in the near UV region. When excited at the appropriate wavelength, this change in the absorbance spectrum translates into a substantial fluorescence turn‐on signal. The described profluorophores are readily conjugated to amino‐modified DNAs and are rapidly uncaged by a triphenylphosphine–DNA probe under the control of a DNA template. In addition, turnover of the probes on the target strand occurs and yields substantial signal amplification.     

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.     

Triplex formation by pyrene-labelled probes for nucleic acid detection in fluorescence assays

Triplex-forming homopyrimidine oligonucleotides containing insertions of a 2'-5' uridine linkage featuring a pyrene moiety at the 3'-position exhibit strong fluorescence enhancement upon binding to double-stranded DNA through Hoogsteen base pairing. It is shown that perfect matching of the new modification to the base pair in the duplex is a prerequisite for strong fluorescence, thus offering the potential to detect single mutations in purine stretches of duplex DNA. The increase in the fluorescence signal was dependent on the thermal stability of the parallel triplex, so a reduction in the pH from 6.0 to 5.0 resulted in an increase in thermal stability from 25.0 to 55.0 degrees C and in an increase in the fluorescence quantum yield (Phi(F)) from 0.061 to 0.179, while the probe alone was fluorescently silent (Phi(F)=0.001-0.004). To achieve higher triplex stability, five nucleobases in a 14-mer sequence were substituted with alpha-L-LNA monomers, which provided a triplex with a T(m) of 49.5 degrees C and a Phi(F) of 0.158 at pH 6.0. Under similar conditions, a Watson-Crick-type duplex formed with the latter probe showed lower fluorescence intensity (Phi(F)=0.081) than for the triplex.     

A PNA-DNA2 Triple-Helix Molecular Switch-Based Colorimetric Sensor for Sensitive and Specific Detection of microRNAs from Cancer Cells

Peptide nucleic acids (PNAs), the synthetic DNA mimics that can bind to oligonucleotides to form duplexes, triplexes, and quadruplexes, could be advantageous as probes for nucleic acid sequences owing to their unique physicochemical and biochemical properties. We have found that a homopurine PNA strand could bind to two homopyrimidine DNA strands to form a PNA-DNA₂ triplex. Moreover, the cyanine dye DiSC₂(5) could bind with high affinity to this triplex and cause a noticeable color change. On the basis of this phenomenon, we have designed a label-free colorimetric sensing platform for miRNAs from cancer cells by using a PNA-DNA₂ triple-helix molecular switch (THMS) and DiSC₂(5). This sensing platform can detect miRNA-21 specifically with a detection limit of 0.18 nM, which is comparable to that of the THMS-mediated fluorescence sensing platform. Moreover, this colorimetric platform does not involve any chemical modification or enzymatic signal amplification, which boosts its applicability and availability at the point of care in resource-limited settings. The universality of this approach can be simply achieved by altering the sequences of the probe DNA for specific targets.     

Fluorogenic Sequencing Using Halogen‐Fluorescein‐Labeled Nucleotides

Fluorogenic sequencing is a sequencing‐by‐synthesis technology that combines the advantages of pyrosequencing and fluorescence detection. With native duplex DNA as the major product, we employ polymerase to incorporate the complement‐ arily matched terminal phosphate‐labeled fluorogenic nucleotides into the DNA template and release halogen‐fluorescein as the reporter. This red‐emitting fluorophore successfully avoids spectral overlap with the autofluorescence background of the flow chip. We fully characterized the enzymatic reaction kinetics of the new substrates, and performed a 35‐base sequencing experiment with 60 reaction cycles. Our achievement expands the substrate repertoire for fluorogenic sequencing, and extends the spectral range to obtain better signal‐to‐background performance.     

Bivalent Display of Dicysteine on Peptide Nucleic Acids for Homogenous DNA/RNA Detection through in Situ Fluorescence Labelling

Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached through covalent bonding to an oligonucleotide probe. However, the additional conjugation step increases costs. Here we introduce a method that avoids the requirement for the preparation of fluorescence‐labelled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine‐containing peptide nucleic acid (PNA) probes to form a bipartite tetracysteine motif that binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases in fluorescence emission (with response factors of up to 80‐fold and high brightness up to 50 mL mol^?1 cm^?1). The detection system provides sub‐nanomolar limits of detection and allows discrimination of single nucleotide variations through more than 20‐fold changes in fluorescence intensity. To demonstrate its usefulness, the FIAsH‐based readout of the bivalent CysCys‐PNA display was interfaced with a rolling‐circle amplification (RCA) assay used to detect disease‐associated microRNA let‐7a.     

Efficient DNA strand displacement by a W-shaped nucleoside analogue (WNA-βT) containing an ortho-methyl-substituted phenyl ring

Molecules that can target duplex DNA with sequence selectivity have the potential to be useful tools in genomic research and also as therapeutic agents. Homopurine-homopyrimidine stretches in duplex DNA can be recognized by homopurine or homopyrimidine TFOs (triplex-forming oligonucleotides) through the formation of triplex DNA. We have previously developed bicyclic nucleoside analogues (WNAs) for the formation of stable triplexes in the formation of stable antiparallel triplexes containing a TA or a CG interrupting site. In this study, we investigated the effects on triplex DNA formation of ortho-, meta-, and para-methyl substituent groups on the aromatic ring of the WNA analogue. It was found that the homopurine TFO containing meta- and para-methyl-substituted WNA-βT (mMe-WNA-βT, pMe-WNA-βT) stabilized triplexes containing a TA interrupting site or a GC site, respectively. Interestingly, the ortho-methyl-substituted WNA-βT (oMe-WNA-βT) efficiently promoted DNA strand displacement to form the TFO/pyrimidine duplex. A detailed investigation showed that the duplex was in the antiparallel orientation and that its formation took place prior to triplex formation with the need for a magnesium cation. NOESY measurements indicated a significant difference in the rotation flexibilities of the phenyl rings of WNA-βTs: that is, the conformation of the ortho-methylated phenyl ring was stable in a temperature-independent manner. It was speculated that the initial formation of a ternary complex was followed by strand displacement and then the formation of the TFO/pyrimidine duplex together with the TFO(2)/pyrimidine triplex formation during the early stage, and that the equilibrium shifted to the triplex during the later stage. Although the detailed role is still uncertain, the fixed phenyl ring of oMe-WNA-βT might play a role in the displacement reaction.     

Forced intercalation probes (FIT Probes): thiazole orange as a fluorescent base in peptide nucleic acids for homogeneous single-nucleotide-polymorphism detection

Fluorescent base analogues in DNA are versatile probes of nucleic acid-nucleic acid and nucleic acid-protein interactions. New peptide nucleic acid (PNA) based probes are described in which the intercalator dye thiazole orange (TO) serves as a base surrogate. The investigation of six TO derivatives revealed that the linker length and the conjugation site decided whether a base surrogate conveys sequence-selective DNA binding and whether fluorescence is increased or decreased upon single-mismatched hybridization. One TO derivative conferred universal PNA-DNA base pairing while maintaining duplex stability and hybridization selectivity. TO fluorescence increased up to 26-fold upon hybridization. In contrast to most other probes, in which fluorescence is invariant once hybridization had occurred, the emission of TO-containing PNA probes is attenuated when forced to intercalate next to a mismatched base pair. The specificity of DNA detection is therefore not limited by the selectivity of probe-target binding and a DNA target can be distinguished from its single-base mutant under nonstringent hybridization conditions. This property should be of advantage for real-time quantitative PCR and nucleic acid detection within living cells.     

An Isocytidine Derivative with a 2‐Amino‐6‐methylpyridine Unit for Selective Recognition of the CG Interrupting Site in an Antiparallel Triplex DNA

Sequence‐specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non‐natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N²‐arylmethyl isodC derivatives and incorporated them into triplex‐forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2‐amino‐6‐methylpyridine moiety (AP‐isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.     

Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells

Classical fluorescence‐based approaches to monitor ligand–protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn‐on probes for a G protein‐coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor‐specific turn‐on response. The new ligand was successfully applied for background‐free imaging and quantification of oxytocin receptors in living cells.     

In Vitro Fluorogenic Real‐Time Assay of the Repair of Oxidative DNA Damage

The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8‐oxoguanine, are a main source of these mutations, and the enzyme 8‐oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8‐oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel‐based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8‐oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8‐oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60‐fold light‐up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays.     

Sequence‐Specific Covalent Capture Coupled with High‐Contrast Nanopore Detection of a Disease‐Derived Nucleic Acid Sequence

Hybridization‐based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence‐specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease‐relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap‐containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast—in essence, digital—signal that enables sensitive, single‐molecule sensing of the cross‐linked duplexes.     

DNA Binding Mode Analysis of a Core-Extended Naphthalene Diimide as a Conformation-Sensitive Fluorescent Probe of G-Quadruplex Structures

G-quadruplex existence was proved in cells by using both antibodies and small molecule fluorescent probes. However, the G-quadruplex probes designed thus far are structure- but not conformation-specific. Recently, a core-extended naphthalene diimide (c_ex-NDI) was designed and found to provide fluorescent signals of markedly different intensities when bound to G-quadruplexes of different conformations or duplexes. Aiming at evaluating how the fluorescence behaviour of this compound is associated with specific binding modes to the different DNA targets, c_ex-NDI was here studied in its interaction with hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex models by biophysical techniques, molecular docking, and biological assays. c_ex-NDI showed different binding modes associated with different amounts of stacking interactions with the three DNA targets. The preferential binding sites were the groove, outer quartet, or intercalative site of the hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex, respectively. Interestingly, our data show that the fluorescence intensity of DNA-bound c_ex-NDI correlates with the amount of stacking interactions formed by the ligand with each DNA target, thus providing the rationale behind the conformation-sensitive properties of c_ex-NDI and supporting its use as a fluorescent probe of G-quadruplex structures. Notably, biological assays proved that c_ex-NDI mainly localizes in the G-quadruplex-rich nuclei of cancer cells.     

Oligonucleotides Containing Phenoxazine Artificial Nucleobases: Triplex-Forming Abilities and Fluorescence Properties

1,3-Diaza-2-oxophenoxazine (“phenoxazine”), a tricyclic cytosine analogue, can strongly bind to guanine moieties and improve π–π stacking effects with adjacent bases in a duplex. Phenoxazine has been widely used for improving duplex-forming abilities. In this study, we have investigated whether phenoxazine and its analogue, 1,3,9-triaza-2-oxophenoxazine (9-TAP), could improve triplex-forming abilities. A triplex-forming oligonucleotide (TFO) incorporating a phenoxazine component was found to show considerably decreased binding affinity with homopurine/homopyrimidine double-stranded DNA, so the phenoxazine system was considered not to function as either a protonated cytosine or thymine analogue. Alternatively, a 9-TAP-containing artificial nucleobase developed by us earlier as a new phenoxazine analogue functioned as a thymine analogue with respect to AT base pairs in a parallel triplex DNA motif. The fluorescence of the 9-TAP moiety was maintained even in triplex (9-TAP:AT) formation, so 9-TAP might be useful as an imaging tool for various oligonucleotide nanotechnologies requiring triplex formation.     

Fluorescence detection of single-nucleotide polymorphism with single-strand triplex-forming DNA probes

Triple-helix-forming oligonucleotides (TFOs) are widespread in the genome and have been found in regulatory regions, especially in promoter zones and recombination hotspots of DNA. To specifically detect these polypurine sequences, we designed and synthesized two dual pyrene-labeled single-strand oligonucleotide probes (TFO-FPs) consisting of recognition, linker, and detection sequences. The hybridization processes of TFO-FPs with target polypurine oligonucleotides involve both Watson-Crick and Hoogsteen base-pairings. Through double sensing of oligonucleotide sequences, single mutations of target oligonucleotides are detected by monitoring changes in pyrene fluorescence. The high specificities of the probes are maintained over a wide temperature range without sacrifice of hybridization kinetics.     

Development of Fluorogenic Probes for Rapid High-Contrast Imaging of Transient Nuclear Localization of Sirtuin 3

Protein labeling using fluorogenic probes enables the facile visualization of proteins of interest. Herein, we report new fluorogenic probes consisting of a rationally designed coumarin ligand for the live-cell fluorogenic labeling of the photoactive yellow protein (PYP)-tag. On the basis of the photochemical mechanisms of coumarin and the probe–tag interactions, we introduced a hydroxy group into an environment-sensitive coumarin ligand to modulate its spectroscopic properties and increase the labeling reaction rate. The resulting probe had a higher labeling reaction rate constant and a greater fluorescence OFF–ON ratio than any previously developed PYP-tag labeling probe. The probe enabled the fluorogenic labeling of intracellular proteins within minutes. Furthermore, we used our probe to investigate the localization of sirtuin 3 (SIRT3), a mitochondrial deacetylase. Although the nuclear localization of SIRT3 has been controversial, this transient nuclear localization was clearly captured by the rapid, high-contrast imaging enabled by our probe.     

A label-free DNA sensor based on impedance spectroscopy

This paper describes a label-free detection system for DNA strands based on gold electrodes and impedance measurements. A single-stranded 18 mer oligonucleotide (ssDNA) was immobilised via a thiol linker on gold film electrodes and served as probe DNA. Residual binding places were filled with mercaptobutanol. The sensor surface clearly distinguished between complementary and non-complementary target ssDNA. Additionally, detection of single base pair mismatches was possible. The electrode was impedimetrically characterised in the presence of the redox system ferri/ferrocyanide before and after DNA hybridisation. Impedance analysis showed that the charge transfer resistance, R_ct, was increasing after DNA duplex formation, whereas the capacitive properties remain rather unaltered. The relative change of R_ct was used as sensor parameter. Concentrations in the nanomolar range have been detected by the system. The sensor was reusable because a denaturation protocol allowed effective double strand dissociation without changing the surface properties of the electrode substantially. The time for DNA detection have been reduced to about 15 min including regeneration. The sensor signal was amplified by about 20% after binding of a negatively charged molecule to the formed DNA duplex. The sensor was also capable of sensing longer target ssDNA strands as shown with 25 mer and 37 mer oligonucleotides.     

Alkoxy Tetrazine Substitution at a Boron Center: A Strategy for Synthesizing Highly Fluorogenic Hydrophilic Probes

Strongly fluorogenic boron dipyrromethene (BODIPY)–tetrazine probes have been obtained by introducing an alkoxy tetrazine fragment at the boron center. The fluorescence signal from these probes strongly increases by up to 225‐fold after reaction with bioorthogonal coupling partners, and the hydrophilicity of probes is improved, such that they are suitable for live‐cell imaging.     

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (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.     

Controlling the Fluorescence of Benzofuran‐Modified Uracil Residues in Oligonucleotides by Triple‐Helix Formation

We developed fluorescent turn‐on probes containing a fluorescent nucleoside, 5‐(benzofuran‐2‐yl)deoxyuridine (dU^BF) or 5‐(3‐methylbenzofuran‐2‐yl)deoxyuridine (dU^MBF), for the detection of single‐stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dU^MBF achieved superior fluorescence enhancement than that containing dU^BF. NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3‐methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3‐methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex‐induced fluorescence enhancement of the oligonucleotide probe containing dU^MBF.