br We have recently shown
We have recently shown that incorporation of a thiazole-or-ange-20-deoxyuridine conjugate, dUTO, 1 (Fig. 1) into an oligonu-cleotide probe targeting cyclin D1 mRNA (a breast cancer marker) allowed the sensitive detection of the target based on a 7-fold enhanced fluorescence of the probe. Fluorescence enhancement was due to hybridization of the ss-20-OMe-RNA-dUTO probe with cyclin D1 mRNA in a total RNA extract from cancerous cells, and subsequent intercalation of the TO moiety into the duplex . However, the signal to background ratio was insufficiently high.
In the present study, we targeted the design of a novel probe for the detection of HER2 biomarker. Amplification or over-expression of this oncogene plays an important role in the development and progression of certain aggressive types of breast cancer. In recent years the HER2 protein has become an important biomarker and target of therapy for approximately 30% of breast cancer patients .
Homo- or heterodimerization of HER receptors results in cell proliferation, survival, differentiation, angiogenesis, and invasion. A healthy breast tissue cell has two copies of the HER2 gene. Some kinds of breast cancer start when a breast tissue cell has more than two copies of that gene, and those copies start over-producing the HER2 protein . As a result, the affected NCT-501 grow and divide far too quickly. The American Cancer Society recommends that all women newly diagnosed with breast cancer get a biopsy test for HER2 . HER2-positive cancer usually occurs in younger women
and is more rapidly-growing and aggressive than other types of breast cancer.
For the hybridization-based detection of HER2 mRNA we have targeted here the implementation of the chromophore of the Green Fluorescent Protein (GFP) as an alternative intercalator to the thiazole-orange moiety in the dUTO (1)-labeled oligonucleotide probe targeting cyclin D1 mRNA . GFP is a naturally fluorescent protein (lem 504 nm; F 0.79; brightness 23 000 M 1cm 1). Its exceptional fluorescence is leveraged as a powerful tool for numerous biochemical and biological applications, including detection of biomolecules within living cells . The GFP fluo-rophore is based on a 4-hydroxybenzylidene imidazolinone (HBI) core, 2 (Fig. 2) . Synthetic HBI analog 3 is non-fluorescent, as is denatured GFP . The source of fluorescence stems from two parts of the p-conjugated system in HBI - the phenol and imida-zolinone groups - interconnected by a methine bridge. Due to the conformational flexibility, the fluorescence of the isolated GFP chromophore in solution is quenched by radiationless internal conversion.
To address the demand for specific, simple, sensitive, and cost-effective nucleic acid detection technologies, and in particular to detect HER-2 mRNA breast cancer marker, we targeted here the development of single-strand (ss)-DNA-nucleoside-intercalator conjugate (DNA-NIC) probe. Specifically, the ssDNA probe cova-lently binds, via a suitable spacer, a duplex-intercalating moiety that has low quantum yield while free in solution. Upon probe hybridization with target mRNA in a total RNA cell extract, the duplex-intercalating moiety undergoes intercalation and exhibits significant F and brightness (Fig. 3), thus indicating the presence of the target mRNA.
Practically, we coupled a derivative of the natural GFP-chromophore to 20-deoxyuridine via a carbon linker, to give 4 (Fig. 4). This modified nucleoside unit was incorporated into a 20-mer ssDNA probe targeting HER-2 mRNA breast cancer marker.
Here, we report on the design and synthesis of 20-deoxyuridine analog, dUHBI, 4, its photophysical properties, the synthesis of dUHBI-labeled 20-mer oligonucleotide hybridization probe (ON1), its photophysical properties in solution vs. upon hybridization with the complementary segment of HER2 mRNA (ON2), and finally, its usefulness for the specific detection of target HER2 mRNA in total RNA extract of cancerous cells.
2. Results and discussion
2.1. Design of 20-deoxyuridine-based Nucleoside Intercalator Conjugate (NIC), 4
We hypothesized, based on the excellent fluorescence of the HBI-chromophore in the rigid environment of naturally fluorescent proteins , that intercalation of dUHBI into a rigid probe-target duplex, will enhance the fluorescence of dUHBI-oligonucleotide probe.
For this purpose, we targeted the synthesis of 4. First, we
Fig. 2. The fluorophore of Green Fluorescent Protein (GFP), 2, and an analog of GFP-
Fig. 1. Thiazole orange-modified 20-deoxy-uridine nucleotide, dUTO, 1. chromophore, 3.
Fig. 3. Schematic representation of the principle of detection of target mRNA using hybridization-probe containing Nucleoside Intercalator Conjugate (NIC).