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European Journal of Medicinal Chemistry
An oligonucleotide probe incorporating the chromophore of green fluorescent protein is useful for the detection of HER-2 mRNA breast cancer marker
Abed Saady a, Verena Bottner€ b, Melissa Meng b, Eli Varon c, Yaron Shav-Tal c, Christian Ducho b, Bilha Fischer a, * a Department of Chemistry, Bar-Ilan University, Ramat-Gan, 52900, Israel
b Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, 66123, Saarbrücken, Germany
c Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat-Gan, 52900, Israel
Received in revised form
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 oligonu-cleotide 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 20-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 (50-CCCGTUTCAACAGGAGTTTC-30), ONHBI, targeting nucleotides 1233e1253 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 inter-calation 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.
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Breast cancer at the molecular level is a heterogeneous disease with distinct entities: luminal A, luminal B, HER2, basal- and normal-like tumors. These entities have different biologic path-ways, cells of origin, clinical features, drug responsiveness and prognoses. Importantly, the molecular profiles of breast cancer are fixed early during breast cancer progression .
To date, expression levels for biomarkers (e.g. Human Epidermal Growth Factor Receptor 2 (HER2), progesterone receptor (PR), and
* Corresponding author.
E-mail address: [email protected] (B. Fischer).
estrogen receptor (ER)) on the protein level, are assessed by immunohistochemical staining, and performed on formalin-fixed biopsy samples. Another approach for detecting and grading cancerous tissues is based on the abnormal mRNA expression levels emanating from unregulated genes . Fluorescent In Situ Hy-bridization (FISH)  technique is used to detect and localize spe-cific DNA regions or mRNA targets in cells and tissue samples. DNA FISH can be performed on breast biopsies to determine whether cells have extra copies of a gene, e.g. HER2 gene, which would imply that the cells have more HER2 receptors. RNA FISH would target the mRNAs of a gene. The RNA FISH methodology, although powerful, requires the fluorescent labeling of the probe with a fluorophore of interest (e.g. FITC or Cy3). This is an expensive technique which requires additional conjugation and purification steps following
probe synthesis. Furthermore, several long oligonucleotides which are multiply-labeled should be prepared for each mRNA target , or alternatively, tens of singly labeled probes . Hence, FISH is not commonly applied in disease characterization.
FISH is considered to be a methodology that only applies to fixed cells, as the use of oligonucleotides in living cells is limited due to their rapid degradation, and the inability to wash away unbound probe leads to high background. These issues have been circum-vented by designing degradation-resistant variants of “molecular beacons” (MBs), which are short oligonucleotide probes with a fluorophore on one end and a quencher on the other. The fluo-rophore and the quencher are in close proximity when the probe is not bound to the target, but are far apart when it is bound to the target; thus, the probe is only fluorescent when bound to the target. MBs reduce the background to the point where one can detect individual mRNA molecules in living cells [6,7]. Upon hybridization of MBs with target mRNA, a fluorescent signal is emitted that can be quantified and correlated with the expression level of the target. Yet, MBs have to be appropriately designed to achieve high sensi-tivity, specificity, nuclease resistance, and long term stability. Another approach for RNA imaging in live cells involves the use of DNA-intercalator stains, such as thiazole orange (TO), which pro-vides a high signal/background ratio. To reduce background fluo-rescence TO was appended to peptide nucleic acid (PNA) via a long flexible linker and was used as a nucleobase surrogate, resulting in improved target specificity. The enforcement of TO intercalation between pre-determined base-pairs upon formation of target-probe duplex provided high brightness .