Hybridization-Sensitive Fluorescent Oligonucleotide Probe Conjugated with a Bulky Module for Compartment-Specific mRNA Monitoring in a Living Cell

Hayashi, Gosuke; Yanase, Masafumi; Takeda, Katsuya; Sakakibara, Daisuke; Sakamoto, Ryosuke; Wang, Dan Ohtan; Okamoto, Akimitsu

doi:10.1021/acs.bioconjchem.5b00090

Cited by 13 publications

(4 citation statements)

References 23 publications

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“…A shortcoming of ECHO probes is their non-specific binding to dsDNA, as well as to each other, due to the intrinsic affinity of the TO dimers to DNA [ 80 , 88 , 89 ]. Attempts to address such problems, however, involved the incorporation of unnatural bases that prevent cross-probe reaction [ 88 ], or the conjugation of the ECHO probes to bulky protein or nanoparticles to avoid diffusion of probes into specific cell organelles [ 90 ]. However, these approaches are limited in solving the issues described above.…”

Section: Sensing Of Nucleic Acidsmentioning

confidence: 99%

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions

Suss,

Motiei,

Margulies

2021

Molecules

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Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable ‘turn-on’ fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules.

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Section: Sensing Of Nucleic Acidsmentioning

confidence: 99%

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions

Suss,

Motiei,

Margulies

2021

Molecules

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“…The long linker allows the TO chromophores to fold back, permitting intercalation and resulting in “dequenching” upon hybridization by resolving the aggregate . ECHO probes have been successfully applied in live-cell RNA imaging . However, covalently linked TO dimers have affinity for double-stranded DNA, which promotes the formation of partially matched probe dimers and therefore increases background emission …”

Section: Fluorogenic Hybridization Probes Based On Thiazole Orangementioning

confidence: 99%

DNA Stains as Surrogate Nucleobases in Fluorogenic Hybridization Probes

Hövelmann

Seitz

2016

Acc. Chem. Res.

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The increasing importance assigned to RNA dynamics in cells and tissues calls for probe molecules that enable fluorescence microscopy imaging in live cells. To achieve this goal, fluorescence dyes are conjugated with oligonucleotides so as to provide strong emission upon hybridization with the target molecule. The impressive 10(3)-fold fluorescence intensification observed when DNA stains such as thiazole orange (TO) interact with double-stranded DNA is intriguing and prompted the exploration of oligonucleotide conjugates. However, nonspecific interactions of DNA stains with polynucleotides tend to increase background, which would affect the contrast achievable in live-cell imaging. This Account describes the development of DNA-stain-labeled hybridization probes that provide high signal-to-background. We focus on our contributions in context with related advances from other laboratories. The emphasis will be on the requirements of RNA imaging in live cells. To reduce background, intercalator dyes such as TO were appended to peptide nucleic acid (PNA), which is less avidly recognized by DNA stains than DNA/RNA. Constraining the TO dye as a nucleobase surrogate in "forced intercalation (FIT) probes" improved the target specificity, presumably by helping to prevent unspecific interactions. The enforcement of TO intercalation between predetermined base pairs upon formation of the probe-target duplex provided for high brightness and enabled match/mismatch selectivity beyond stringency of hybridization. We show examples that highlight the use of PNA FIT probes in the imaging of mRNA, miRNA, and lncRNA in living cells. The "FIT approach" was recently extended to DNA probes. Signal brightness can become limiting when low-abundance targets ought to be visualized over cellular autofluorescence. We discuss strategies that further the brightness of signaling by FIT probes. Multilabeling with identical dyes does not solve the brightness issue. To avoid self-quenching, we combined two different yet spectrally overlapping fluorescent base surrogates. A hybridization-sensitive dye serves as a light collector that transfers energy to a brightly emissive acceptor dye. To improve the brilliance of single-dye probes, the "TO-nucleotide" was accompanied by an adjacent locked nucleic acid (LNA) unit. The LNA-constrained FIT probes are responsive and bright, enabling the tracking of mRNA transport in living tissue. We also show that the color repertoire of FIT probes is not restricted to the green-emissive TO but can be expanded to cyan and red. A new base surrogate (4,4-linked bisquinoline) provided up to 195-fold enhancement of the fluorescence.

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“…8 Attachment of fluorophores directly to LNA or to other scaffolds within LNA/DNA probes has an additional positive effect on SNP sensitivity. 8 In the present work we combine recent findings by us and others on 'clickable' LNA/DNA probes, 8,9 conditionally fluorescent oligonucleotides [10][11][12][13] and a solid-support hybridization assay, aiming at the development of an ultra-specific and sensitive probes for cancer DNA detection. 14 Herein, we introduce a series of fluorescent probes containing internally positioned PAH and xanthene dyes leading simultaneously to high binding affinity and efficient recognition of BRAF and KRAS oncogenes ( Fig.…”

mentioning

confidence: 98%

Environmentally sensitive molecular probes reveal mutations and epigenetic 5-methyl cytosine in human oncogenes

2017

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There is currently an unmet need for reliable tools that allow for direct detection and quantification of modifications in genomic DNA. For example, in cancer research and clinical diagnostics, target DNA has to be amplified and sequenced in order to reveal mutations. For 5-methylcytosine detection, bisulfite treatment of DNA is applied for the analysis, which often leads to poor specificity and reproducibility of the results. Herein we describe a simple approach that specifically detects clinically significant modifications in the human oncogenes BRAF and KRAS. We prove that this can be done using a fast and reliable hybridization assay applying novel internally labelled oligonucleotide probes and optical detection methods.

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Hybridization-Sensitive Fluorescent Oligonucleotide Probe Conjugated with a Bulky Module for Compartment-Specific mRNA Monitoring in a Living Cell

Cited by 13 publications

References 23 publications

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions

DNA Stains as Surrogate Nucleobases in Fluorogenic Hybridization Probes

Environmentally sensitive molecular probes reveal mutations and epigenetic 5-methyl cytosine in human oncogenes

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