5'-fluoro(di)phosphate-labeled oligonucleotides are versatile molecular probes for studying nucleic acid secondary structure and interactions by 19F NMR.

The high sensitivity of 19F nucleus to changes in the chemical environment has promoted the use of fluorine-labeled molecular probes to study structure and interactions of nucleic acids by 19F NMR. So far, most efforts have focused on incorporating the fluorine atom into nucleobase and ribose moieties using either monomer building blocks for solid-phase synthesis, or nucleoside triphosphates for enzymatic synthesis. Here, we report a simple and efficient synthesis of 5'-fluoromonophosphorylated and 5'-fluorodiphosphorylated oligodeoxyribonucleotides, which combines solid-phase and in-solution synthesis methods and requires only commercially available nucleoside phosphoramidites, followed by their evaluation as 19F NMR probes. We confirmed that the fluorine atom at the oligonucleotide 5' end did not alter the secondary structure of DNA fragments. Moreover, at the same time, it enabled real-time 19F NMR monitoring of various DNA-related biophysical processes, such as oligonucleotide hybridization (including mismatch identification), G-quadruplex folding/unfolding and its interactions with thrombin, as well as formation of an i-motif structure and its interaction with small-molecule ligands.

Live-Cell Single RNA Imaging Reveals Bursts of Translational Frameshifting.

Ribosomal frameshifting during the translation of RNA is implicated in human disease and viral infection. While previous work has uncovered many details about single RNA frameshifting kinetics in vitro, little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames. Applying this technology to RNA encoding the HIV-1 frameshift sequence reveals a small subset (∼8%) of the translating pool robustly frameshift. Frameshifting RNA are translated at similar rates as non-frameshifting RNA (∼3 aa/s) and can continuously frameshift for more than four rounds of translation. Fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide insight into retroviral frameshifting and could lead to alternative strategies to perturb the process in living cells.

Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting.

We have synthesised a range of thiazole orange (TO) functionalised oligonucleotides for nucleic acid detection in which TO is attached to the nucleobase or sugar of thymidine. The properties of duplexes between TO-probes and their DNA and RNA targets strongly depend on the length of the linker between TO and the oligonucleotide, the position of attachment of TO to the nucleotide (major or minor groove) and the mode of attachment of thiazole orange (via benzothiazole or quinoline moiety). This information can be used to design probes for detection of target nucleic acids by fluorescence or duplex melting. With cellular imaging in mind we show that 2'-OMe RNA probes with TO at the 5-position of uracil or the 2'-position of the ribose sugar are particularly effective, exhibiting up to 44-fold fluorescence enhancement against DNA and RNA, and high duplex stability. Excellent mismatch discrimination is achieved when the mispaired base is located adjacent to the TO-modified nucleotide rather than opposite to it. The simple design, ease of synthesis and favourable properties of these TO probes suggest applications in fluorescent imaging of DNA and RNA in a cellular context.

An oligonucleotide probe incorporating the chromophore of green fluorescent protein is useful for the detection of HER-2 mRNA breast cancer marker.

Diagnosis and treatment of breast cancer can be greatly enhanced and personalized based on the quantitative detection of mRNA markers. Here, we targeted the development of a fluorescent oligonucleotide probe to detect specifically the HER-2 mRNA breast cancer marker. We have selected the chromophore of the Green Fluorescent Protein (GFP), 4-hydroxybenzylidene imidazolinone (HBI), as a fluorophore covalently bound to an oligonucleotide probe and potentially capable of intercalating within a probe-mRNA duplex. We first synthesized the two-ring scaffold of the HBI chromophore 5 and coupled it to 2'-deoxyuridine at C5-position via a 7-atom-spacer, to give 4. Indeed, in the highly viscous glycerol used to mimic the reduced conformational flexibility of the intercalated HBI, chromophore 4 displayed a quantum yield of 0.29 and brightness of 20600 M-1cm-1, while no fluorescent signal was observed in methanol. Next, we synthesized a 20-mer oligonucleotide probe incorporating 4 at position 6 (5'-CCCGTUTCAACAGGAGTTTC-3'), ONHBI, targeting nucleotides 1233-1253 of HER-2 mRNA. A 16-fold enhancement of ONHBI emission intensity upon hybridization with the complementary RNA vs that of the oligonucleotide probe alone indicated the presence of target oligonucleotide and proved the intercalation of the chromophore (quantum yield 0.52; brightness 23500 M-1cm-1). Even more, an 11-fold enhancement of ONHBI emission (quantum yield 0.50; brightness 23200 M-1cm-1) was observed when the probe was mixed with total RNA extract from a human cell line that has high levels of HER2 mRNA expression. Thus, we propose ONHBI as a promising probe potentially useful for the sensitive and specific detection of HER2 mRNA breast cancer marker.

Fluorescence in situ hybridization in plants: recent developments and future applications.

Fluorescence in situ hybridization (FISH) was developed more than 30 years ago and has been the most paradigm-changing technique in cytogenetic research. FISH has been used to answer questions related to structure, mutation, and evolution of not only individual chromosomes but also entire genomes. FISH has served as an important tool for chromosome identification in many plant species. This review intends to summarize and discuss key technical development and applications of FISH in plants since 2006. The most significant recent advance of FISH is the development and application of probes based on synthetic oligonucleotides (oligos). Oligos specific to a repetitive DNA sequence, to a specific chromosomal region, or to an entire chromosome can be computationally identified, synthesized in parallel, and fluorescently labeled. Oligo probes designed from conserved DNA sequences from one species can be used among genetically related species, allowing comparative cytogenetic mapping of these species. The advances with synthetic oligo probes will significantly expand the applications of FISH especially in non-model plant species. Recent achievements and future applications of FISH and oligo-FISH are discussed.

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay.

Nucleic acid hybridisation plays a key role in many biological processes, including transcription, translation, and regulation of gene expression. Several sophisticated applications rely on this fundamental interaction, including the polymerase chain reaction, sequencing, and gene therapy. To target a nucleic acid sequence specifically, synthetic oligonucleotides with a suitable affinity and specificity towards the target need to be designed. The affinity of potential probes or therapeutic agents to their target sequence is generally investigated by melting experiments, which break the hydrogen-bonding and stacking interactions that stabilise the double helix resulting in the formation of two single strands. In this paper, we report a comparative study of hybridisation for short fluorescent oligonucleotides labelled with cyanine and ATTO dyes, performed by the currently used UV melting assay and by a more sensitive fluorescence melting experiment. Using different oligonucleotide sequences in the concentration range of 5 nm to 2 µm, we observed a stabilising effect of the fluorophores on the duplexes, especially at low concentrations. We paid particular attention to the effect of polycations and to molecular crowding as major parameters that define the stability and the geometry of nucleic acid duplexes in biological samples. We also demonstrated how the fluorometry-based melting data could aid the design of a probe targeting a human BRAF gene fragment to reduce the off-target binding by a factor of seven.

Design and synthesis of novel photoinduced electron transfer-based hybridization probes.

Photoinduced electron transfer (PeT)-based hybridization probe is a linear and quencher-free oligonucleotide (ON) probe for DNA or RNA detection. In this report, we designed and synthesized novel adenosine analogues for PeT-based hybridization probe. In particular, the analogue containing a piperazinomethyl moiety showed effective quenching property under physiological conditions. When the probe containing the analogue was hybridized with a complementary DNA or RNA, the fluorescence increased 3- or 4-fold, respectively, compared to the single-stranded state.

An in-house multilocus SNP genotyping assay for evaluation of complex genetic diseases.

BACKGROUND: With an increase in the discovery of newer genetic loci/polymorphisms in complex multifactorial diseases, there is also an increased need for methods that can simultaneously genotype multiple loci in a cost-effective manner. Using coronary artery disease (CAD) as a model, the study aimed to develop an in-house multilocus assay for simultaneous detection of 17 genetic variants in 11 genes implicated in CAD. METHODS: A multiplex polymerase chain reaction (PCR)-based reverse line blot hybridization (MPCR-RLBH) approach was used, where each DNA sample was amplified using two separate MPCRs, and the alleles were genotyped using covalently immobilized, amino-linked sequence-specific oligonucleotide probes using an enhanced chemiluminescence system. The assay performance was tested on 75 healthy controls and 75 angiographically proven CAD cases. Validation was done by automated Sanger sequencing. RESULTS: The assay could successfully discriminate both the alleles at CETP (I405V), LPL (D9N), NOS3 (T-786G and E298D), LIPC (C-514T), FGB (G-455A), ITGB3 (L33P), AGT (M235T), and MTR (A2756G) loci. Certain mutations included in this assay such as ins242G, ins397G, E387K, L393K in the LDLR; N291S in the LPL; D442G in the CETP; and T833C in the CBS genes were found to be absent. The genotype results obtained using this assay showed 100% concordance with sequencing. CONCLUSION: The study demonstrated development and validation of a multiplex SNP genotyping assay that can be used to assess genetic risk factors in CAD. The assay provides a cost-effective alternative to expensive high throughput genotyping systems in common molecular research laboratories.

6-Aryl-4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (benzo-fused 7-deaza-dATP analogues): Synthesis, fluorescent properties, enzymatic incorporation into DNA and DNA-protein binding study.

Four 6-substituted 4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (dA(BX)TPs) were prepared by glycosylation of 4,6-dichloropyrimidoindole followed by ammonolysis, cross-coupling and triphosphorylation. They were found to be moderate to good substrates for DNA polymerases in primer extension. They also exerted fluorescence with emission maxima 335-378nm. When incorporated to oligonucleotide probes, they did not show significant mismatch discrimination but the 6-benzofuryl 4-amino-pyrimido[4,5-b]indole nucleotide displayed a useful sensitivity to protein binding in experiment with SSB protein.

Microenvironmental Effect of 2'-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA.

2'-O-(1-Pyrenylmethyl)uridine modified oligoribonucleotides provide highly sensitive pyrene fluorescent probes for detecting specific nucleotide mutation of RNA targets. To develop more stable and cost-effective oligonucleotide probes, we investigated the local microenvironmental effects of nearby nucleobases on pyrene fluorescence in duplexes of RNAs and 2'-O-(1-pyrenylmethyl)uridine modified oligonucleotides. By incorporation of deoxyribonucleotides, ribonucleotides, 2'-MeO-nucleotides and 2'-F-nucleotides at both sides of 2'-O-(1-pyrenylmethyl)uridine (U(p)) in oligodeoxynucleotide probes, we synthesized a series of pyrene modified oligonucleotide probes. Their pyrene fluorescence emission spectra indicated that only two proximal nucleotides have a substantial effect on the pyrene fluorescence properties of these oligonucleotide probes hybridized with target RNA with an order of fluorescence sensitivity of 2'-F-nucleotides > 2'-MeO-nucleotides > ribonucleotides ≫ deoxyribonucleotides. While based on circular dichroism spectra, overall helix conformations (either A- or B-form) of the duplexes have marginal effects on the sensitivity of the probes. Instead, the local substitution reflected the propensity of the nucleotide sugar ring to adopt North type conformation and, accordingly, shifted their helix geometry toward a more A-type like conformation in local microenvironments. Thus, higher enhancement of pyrene fluorescence emission favored local A-type helix structures and more polar and hydrophobic environments (F > MeO > OH at 2' substitution) of duplex minor grooves of probes with the target RNA. Further dynamic simulation revealed that local microenvironmental effect of 2'-F-nucleotides or ribonucleotides was enough for pyrene moiety to move out of nucleobases to the minor groove of duplexes; in addition, 2'-F-nucleotide had less effect on π-stack of pyrene-modified uridine with upstream and downstream nucleobases. The present oligonucleotide probes successfully distinguished target RNA from single-mutated RNA analyte during an in vitro assay of RNA synthesis.

Simultaneous RNA-DNA FISH.

A highly useful tool for studying lncRNAs is simultaneous RNA-DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA-DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA.

Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs.

MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking-to-unlocking system is based on fold-back anchored DNA templates that consist of a cytosine-rich loop for AgNCs stabilization, an miRNA recognition site and an overlap region for hairpin stabilization. When an miRNA is recognized, fluorescence in the visible region is specifically extinguished in a concentration-dependent manner. Here, the exact composition of the fold-back anchor for the locking-to-unlocking system has been systematically optimized, balancing propensity for loop-structure formation, encapsulation of emissive AgNCs and target sensitivity. It is demonstrated that the applied strategy successfully can detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines.

Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs.

Nucleic acid hybridization probes are sought after for numerous assay and imaging applications. These probes are often limited by the properties of fluorescent dyes, prompting the development of new probes where dyes are paired with novel or nontraditional luminescent materials. Luminescent terbium complexes are an example of such a material, and these complexes offer several unique spectroscopic advantages. Here, we demonstrate two nonstem-loop designs for light-up nucleic acid hybridization beacons that utilize time-resolved Förster resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved emission from the dye provides an analytical signal. Both designs are based on probe oligonucleotides that are labeled at their opposite termini with Tb and a fluorescent reporter dye. In one design, a probe is partially blocked with a quencher dye-labeled oligonucleotide, and target hybridization is signaled through toehold-mediated strand displacement and loss of a competitive FRET pathway. In the other design, the intrinsic folding properties of an unblocked probe are utilized in combination with a temporal mechanism for signaling target hybridization. This temporal mechanism is based on a recently elucidated "sweet spot" for TR-FRET measurements and exploits distance control over FRET efficiencies to shift the Tb lifetime within or outside the time-gated detection window for measurements. Both the blocked and unblocked beacons offer nanomolar (femtomole) detection limits, response times on the order of minutes, multiplexing through the use of different reporter dyes, and detection in complex matrices such as serum and blood. The blocked beacons offer better mismatch selectivity, whereas the unblocked beacons are simpler in design. The temporal mechanism of signaling utilized with the unblocked beacons also plays a significant role with the blocked beacons and represents a new and effective strategy for developing FRET probes for bioassays.

Solid- and solution-phase synthesis and application of R6G dual-labeled oligonucleotide probes.

A novel N-TFA-protected carboxyrhodamine 6G (R6G) phosphoramidite was synthesized for use in an automated DNA synthesis to prepare 5'-labeled oligonucleotides. Deprotection and purification conditions were optimized for 5'-labeled and dual-labeled oligonucleotide probes. As an alternative we synthesized an azide derivative of R6G for CuAAC post-synthetic oligonucleotide labeling. Dual-labeled probes obtained by both methods showed the same efficacy in a quantitative PCR assay. R6G-labeled probes demonstrated superior properties in a qPCR assay in comparison with alternative HEX, JOE and SIMA dyes due to more efficient fluorescence quenching by BHQ-1. We successfully used R6G dual-labeled probes for rotavirus genotyping.

ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.

Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

High-resolution and specific detection of bacteria on complex surfaces using nanoparticle probes and electron microscopy.

The study of the interaction of bacteria with surfaces requires the detection of specific bacterial groups with high spatial resolution. Here, we describe a method to rapidly and efficiently add nanogold particles to oligonucleotide probes, which target bacterial ribosomal RNA. These nanogold-labeled probes are then used in an in situ hybridization procedure that ensures both cellular integrity and high specificity. Electron microscopy subsequently enables the visualization of specific cells with high local precision on complex surface structures. This method will contribute to an increased understanding of how bacteria interact with surface structures on a sub-micron scale.

New developments in comet-FISH.

The comet assay combined with fluorescence in-situ hybridisation (FISH) is a powerful technique for comparative analyses of damage induction and repair in genomes and in specific DNA sequences within single cells. Recent advances in the methodology of comet-FISH will be considered here, with particular attention to the design and generation of fluorescent probes. In general, all the approaches must fulfil a few basic requirements: the probes should be no longer than ~300 nucleotides in length (single or double stranded) to be able to penetrate the gel in which the target genomic DNA is embedded, they should be sequence-specific, and their signal should be detectable and distinct from the background fluorescence and the dye used to stain the DNA.

Development of the excimer probe responsible for DNA target bearing the silylated pyrenes at base moiety.

For the development of the excimer probe responsible for DNA target, the deoxyuridine phosphoramidite derivative bearing the silylated pyrene attached at the C-5 position was prepared and incorporated into oligonucleotides. The modified oligonucleotides showed the excimer emission in the absence of the target DNA, on the other hands, the excimer emission was quenched in the presence of the target DNA. For the utilization of the fluorescence behavior, the novel molecular beacon probe containing the silylated pyrene-modified nucleoside at the stem region was designed and the fluorescence property of the probe found to show the responsibility for DNA target.

Using amino-labeled nucleotide probes for simultaneous single molecule RNA-DNA FISH.

Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts.