Development of novel fluorescent probes for molecular diagnosis of breast cancer, targeting personal
Currently, molecular diagnostics of breast cancer is still sub-optimal. The use of newer methods that can refine treatment decision is highly expensive. The avoidance of use of these method due to its high cost results sometimes in wrong treatment. Therefore, there is an urgent need in a specific, simple, fast, sensitive, and cost-effective technology for accurate diagnosis of breast cancer for tailored therapy for individual patients. Our objective is molecular diagnosis of breast cancer subtype. Specifically, we use of tissue- and tumor-specific genes as diagnostic biomarkers, applying our recently developed fluorescent probes, for defining the treatment course for breast cancer patients, as well as enabling post-operative monitoring of patients.
We developed novel fluorescent probes based on nucleosides bearing an intercalating moiety (NIC analogues, e.g. thiazole orange conjugated via a linker to deoxy uridine, dUTO) used for incorporation into 2’-OMe-RNA sequences to form NIC probes. One of the NIC probes targeting cyclin D1 mRNA detected specifically and successfully a minute amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells. Furthermore, this NIC-probe could detect cyclin D1 mRNA target only at high concentrations. Hence, dUTO oligonucleotides are useful hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.
Next, we developed nucleosides containing a flexible spacer with an intercalating moiety at its end (NIC molecules) based on 4-hydroxybenzylidene imidazolinone (HBI), found in the Green Fluorescent Protein. We synthesized 20-mer oligonucleotides incorporating dUHBI, and the corresponding dUDFHBI. These oligonucleotides target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of the probes with complementary 2′-OMe-RNA resulted in 46-fold increase in fluorescence intensity. CD and 19F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes. Furthermore, addition of the probes to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement. The latter probes sensitively detected low concentrations of the target mRNA. These probes were photo-stable for 200 min. In addition, we quantified the number of HER-2 transcripts in a cell. These diagnostic probes are useful 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.
Detection of Cyclin D1 mRNA by hybridization sensitive NIC - oligonucleotide probe. M. Kovaliov, M. Segal, P. Kafri, E. Yavin, Y. Shav-Tal and B. Fischer. Bioorg. Med. Chem. 2014, 22, 2613-2621.
An oligonucleotide probe incorporating the chromophore of green fluorescent protein is useful for the detection
of HER-2 mRNA breast cancer marker. A. Saady, M. Meng, V. Böttner, E. Varon, Y. Shav-Tal, C. Ducho, and B. Fischer.
Eur. J. Med. Chem. 2019,173, 99-106.
Specific, Sensitive, and Quantitative Detection of HER-2 mRNA Breast Cancer Marker by Fluorescent Light-Up
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