Detection of mRNA of the Cyclin D1 Breast Cancer Marker by a Novel Duplex-DNA Probe

Segal, Meirav; Yavin, Eylon; Kafri, Pinhas; Shav‐Tal, Yaron; Fischer, Bilha

doi:10.1021/jm301838y

Cited by 14 publications

(6 citation statements)

References 48 publications

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“…In this context, fluorescence spectroscopy is widely used in nucleic acids research to study the structure and the dynamics as well as the kinetics of protein-DNA interactions [1]. Different fluorescent analogues of DNA bases have been evaluated for examining of DNA conformational transitions [2]–[8]. Most of them are sensitive to the quenching of DNA nucleobases.…”

Section: Introductionmentioning

confidence: 99%

New Environment-Sensitive Multichannel DNA Fluorescent Label for Investigation of the Protein-DNA Interactions

et al. 2014

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Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5′-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.

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Section: Introductionmentioning

confidence: 99%

New Environment-Sensitive Multichannel DNA Fluorescent Label for Investigation of the Protein-DNA Interactions

et al. 2014

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“…HBI-analogue 11 was coupled to dU also via a spacer of 7 atoms (Scheme 2). Specifically, we coupled a 6-atom spacer at the dU C5-position by Suzuki reaction of 5-iodo-2'-deoxyuridine with (E)-6-chloro-1-hexenylboronic acid pinacol ester to get 15 at 83% yield [17], followed by S N 2 reaction with sodium azide to obtain 16. The latter, without purification, was reacted with triphenylphosphine to give 17 at 79% yield [18].…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Photophysical Properties of Conjugates of Green Fluorescent Protein (GFP) Chromophore and 2'-Deoxy-Uridine Developed as Labelled Building Blocks for Oligonucleotide Probes

Saady¹,

Fischer²

2019

JPP

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Fluorescent probes with high signal/background ratio are needed for hybridization assays. Hence, the chromophore of green fluorescent protein (GFP), 4-hydroxybenzylidene-imidazolinone (HBI), was targeted as a potential fluorescent intercalator. For producing a building block for fluorescent oligonucleotide probes, 2'-deoxyuridine (dU) was conjugated with HBI via a flexible carbon-spacer. dU HBI conjugates highly fluoresce in glycerol (λ em 460 nm, Φ 0.31), and are 109-fold more emissive than in methanol, implying the potential of dU HBI-labeled oligonucleotides as probes for hybridization assays.

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“…Breast cancer is a heterogeneous disease. Diagnosis of breast cancer can be greatly enhanced and personalized based on the specific detection of mRNA markers. − Nucleosides containing an intercalating moiety (NIC) represent an innovative approach for the detection of genetic material. , For example, we detected cyclin D1 mRNA in total RNA extract from cancer cells through this methodology, using 5-( trans - N -(hexen-1-yl-)-thiazolium orange)-uridine, dU TO , 1 (Figure ), which was incorporated into an oligonucleotide targeting cyclin D1 mRNA. The detection of the target was determined by the increase in fluorescence signal.…”

Section: Introductionmentioning

confidence: 99%

Specific, Sensitive, and Quantitative Detection of HER-2 mRNA Breast Cancer Marker by Fluorescent Light-Up Hybridization Probes

et al. 2020

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Currently, there is demand for fluorescent oligonucleotide probes for diagnostic purposes. To address this necessity, we developed nucleosides containing a flexible spacer with an intercalating moiety at its end (NIC molecules). The intercalator is based on 4-hydroxybenzylidene imidazolinone (HBI), found in the Green Fluorescent Protein. We synthesized 20-mer oligonucleotides, ON1−ON4, incorporating the DMTr phosphorodiamidite monomer of dU HBI , 2, and the corresponding dU DFHBI , 5b, monomer. ON1−ON4 target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of ON1/ON2 and ON3/ON4 with complementary 2′-OMe-RNA resulted in emission at 462 and 481 nm, respectively, and up to 46-fold increase in fluorescence intensity. CD and 19 F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes (up to ΔT m 6 °C). Furthermore, addition of ON1−ON4 to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement of ON3 and ON4. The latter sensitively detected low concentrations of the target mRNA (at total RNA 30 ng/μL). These probes were photostable for 200 min. Using a dilution curve, we quantified the number of HER-2 transcripts in a cell. In conclusion, ON3 and ON4 are promising diagnostic probes for an easy, instantaneous, specific, and sensitive detection of levels of oncogenes. Importantly, the NIC concept, demonstrated here for diagnostics of breast cancer, is universal and may be applied not only in a clinical setting but also for the detection of any RNA.

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Detection of mRNA of the Cyclin D1 Breast Cancer Marker by a Novel Duplex-DNA Probe

Cited by 14 publications

References 48 publications

New Environment-Sensitive Multichannel DNA Fluorescent Label for Investigation of the Protein-DNA Interactions

New Environment-Sensitive Multichannel DNA Fluorescent Label for Investigation of the Protein-DNA Interactions

Synthesis and Photophysical Properties of Conjugates of Green Fluorescent Protein (GFP) Chromophore and 2'-Deoxy-Uridine Developed as Labelled Building Blocks for Oligonucleotide Probes

Specific, Sensitive, and Quantitative Detection of HER-2 mRNA Breast Cancer Marker by Fluorescent Light-Up Hybridization Probes

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