br Fluorescence properties of dUHBI labeled oligonucleotide
2.5. Fluorescence properties of dUHBI-labeled oligonucleotide probe.
Our main interest pertains to the detection of mRNA with its complementary probe. Therefore, we first investigated the photo-physical properties of dUHBI-labeled oligonucleotide upon hybrid-ization to a specific target in homogeneous solution. We compared the photophysical properties of dUHBI-labeled ssON1 (50-CCCGTUTCAACAGGAGTTTC-30); the complementary 20-OMe-RNA (a segment of HER-2), ssON2 (30-GGGCAAAGTTGTCCTCAAAG-50);
and dsDNA: 20-OMe-RNA, dsON3 (Figs. 5e6). Emission spectra of ON1, ONHBI, and ON2 in PBS buffer upon excitation at 390 nm, at room temperature, showed a very weak emission. However, upon hybridization of ssON1 with complementary 20-OMe-RNA, ssON2, a 16-fold increase in fluorescence was observed vs. that of the oligonucleotide probe alone (Fig. 6). This fluorescence
enhancement indicated the presence of target oligonucleotide and proved the intercalation of the chromophore (quantum yield 0.52; brightness 23500 M 1cm 1). We applied ON1 (ONHBI) to detect HER-2 mRNA in total RNA extracted from two control cell lines which do not express HER2 vs. a cell line overexpressing the HER2 mRNA. Control Ethylmalonyl Coenzyme A were the human U2OS osteosarcoma cell line (HER2 negative), and CAMA-1 cells (a luminal-type human breast cancer cell line; HER2-negative and estrogen-receptor/progesterone-receptor (ER/PR)-positive). The HER2 positive cell line were ZR-75-30 cells (human breast carcinoma, high gene expression of HER2). Specifically, the fluorescence of the ONHBI probe was measured, before and after its addition to RNA cell extract from these cells. ONHBI in PBS buffer exhibited no fluorescence. After 10 min RNA extract containing high levels of HER2 was added. We measured our control by the fluorescence intensity of RNA extract alone (Fig. 7), and of extracts of cells that express basal level of HER2. The emission intensity of ONHBI increased almost immediately (1 min) upon addition of RNA extracts at room temperature (Fig. 7), indicating a rapid hybridi-zation reaction between single stranded ONHBI probe and the target mRNA. We preformed this experiment in duplicates and on two different extract batches on three separate days using the same concentrations of ONHBI probe and RNA extracts.
Upon hybridization, fluorescence enhancement of 11-fold was observed for RNA extracted from the HER2-overexpressing cells as compared to those that do not express. These results demonstrate qualitatively the efficiency of ONHBI for the selective detection of a breast cancer marker, HER2 mRNA, in total RNA extracted from
Fig. 7. Fluorescence spectra of ONHBI probe in the presence of total RNA cell extract containing high levels of HER-2 mRNA and controls. Conditions: 0.3 mM probe in 100 mL PBS buffer (pH 7.4, 25 C), and 179 ng/mL (after addition) total RNA extract, lex 390 nm.
The photophysical properties of the intercalated HBI in probe-mRNA duplex (labs 388, lem 486 and F 0.52) are similar to those of the native green fluorescent protein (labs 395, lem 510 and F 0.79). The differences between our data and the properties of the HBI within the protein are due to the different environment sur-rounding the intercalator moiety as well as the presence of the para-methoxy group (OCH3) rather than para-hydroxyl (OH) at the HBI-aromatic ring in ONHBI and GFP, respectively. We used here the methoxy, rather than the hydroxyl, group for synthetic reasons.
In the light of the above data, we propose an oligonucleotide incorporating dUHBI, ONHBI, as a promising hybridization-probe potentially useful for the sensitive and specific detection of HER2 mRNA breast cancer marker.
4. Experimental section
In order to inspect the sensitivity of the probe, we used a
different concentration series of the total RNA extracted (100, 50, 30, 15 and 10 ng/mL) with ONHBI. We found that the fluorescence
signal depends on the concentration of the total RNA extracted, and the fluorescent signal decreases as concentration decreases (Fig. 8). At concentrations of 100 and 50 ng/mL a fluorescent signal was detected.
In conclusion, we have designed a hybridization oligonucleotide probe incorporating dUHBI, ONHBI. Fluorescence enhancement of 11-fold was observed upon addition of ONHBI-probe to total RNA extracted from the HER2-overexpressing cells as compared to those that do not express. The hybridization-sensitive, quencher-free fluorescent probe, ONHBI, proved to be applicable for the specific and sensitive detection of HER2 mRNA at low total RNA concen-tration (down to 50 ng/mL).