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

Saady, Abed; Böttner, Verena; Meng, Melissa; Varon, Eli; Shav‐Tal, Yaron; Ducho, Christian; Fischer, Bilha

doi:10.1016/j.ejmech.2019.04.013

Cited by 12 publications

(18 citation statements)

References 16 publications

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“…The photophysical properties of compounds 6 and 7 were evaluated in various solvents at a range of viscosities which was reported in details in previous study [9]. UV spectra of compounds 6 and 7 in all solvents exhibited λ abs 386 nm and 20,600 M -1 •cm -1 .…”

Section: Resultsmentioning

confidence: 95%

“…3), into an oligonucleotide probe targeting HER2 mRNA (a breast cancer marker), allowed the sensitive detection of the target based on an 11-fold enhanced fluorescence of the probe. Fluorescence enhancement was due to hybridization of the ss-2'-OMe-RNA-dU HBI probe with HER2 mRNA in total RNA extract from cancerous cells, and subsequent intercalation of the HBI moiety [9]. The length of the spacer that connects to HBI and 2'-deoxyuridine was not investigated.…”

Section: Introduction mentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 95%

Section: Introduction mentioning

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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“…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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“…5 As a fact, studies in biology, physiology, development, pharmacokinetics, oncology and botany have benefited tremendously from the applications of GFP and other fluorescent proteins. [6][7][8][9][10][11][12][13] Furthermore, in vaccine research and development, GFP was incorporated into vector constructions to facilitate the recognition of recombinants. 14 In radiation biology studies, introduction of GFP as a reporter gene into mammalian cells allows visualization of the modified gene expression levels, signal transduction rates, cell metabolism activities, cell cycle phases and cell death, supplying basic information on the cellular response to ionizing radiation (IR).…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Altered Response to Total Body Irradiation of C57BL/6-Tg (CAG-EGFP) Mice

et al. 2020

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Application of green fluorescent protein (GFP) in a variety of biosystems as a unique bioindicator or biomarker has revolutionized biological research and made groundbreaking achievements, while increasing evidence has shown alterations in biological properties and physiological functions of the cells and animals overexpressing transgenic GFP. In this work, response to total body irradiation (TBI) was comparatively studied in GFP transgenic C57BL/6-Tg (CAG-EGFP) mice and C57BL/6 N wild type mice. It was demonstrated that GFP transgenic mice were more sensitive to radiation-induced bone marrow death, and no adaptive response could be induced. In the nucleated bone marrow cells of GFP transgenic mice exposed to a middle dose, there was a significant increase in both the percentage of cells expressing pro-apoptotic gene Bax and apoptotic cell death. While in wild type cells, lower expression of pro-apoptotic gene Bax and higher expression of anti-apoptotic gene Bcl-2, and significant lower induction of apoptosis were observed compared to GFP transgenic cells. Results suggest that presence of GFP could alter response to TBI at whole body, cellular and molecular levels in mice. These findings indicate that there could be a major influence on the interpretation of the results obtained in GFP transgenic mice.

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

Cited by 12 publications

References 16 publications

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

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

Altered Response to Total Body Irradiation of C57BL/6-Tg (CAG-EGFP) Mice

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