Specific, Sensitive, and Quantitative Detection of HER-2 mRNA Breast Cancer Marker by Fluorescent Light-Up Hybridization Probes

Abstract

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.

Environment-Sensitive Nucleoside Probe Unravels the Complex Structural Dynamics of i-Motif DNAs

“…Therefore, we envisioned to integrate two powerful readouts in a single nucleoside probe, namely, fluorescence and 19 F NMR, for the following reasons. Conjugating a heterocycle to a nucleobase can produce environment-sensitive fluorescent nucleoside analogues. − A few such analogues serve as good probes to investigate noncanonical nucleic acid structures, especially GQs. − On the other hand, the 19 F atom, due to high natural abundance, sensitivity to the environment, and “zero” background signal from cells, is emerging as a very useful biophysical label to probe nucleic acid secondary structures and their interaction with ligands and proteins. − We combined these two features and developed fluorobenzofuran-modified nucleosides, which served as good tools to probe GQs of human telomeric overhang . However, this modification is not ideally suited for capturing lowly populated structures as it requires high concentrations of the ON sample.…”

Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives

“…A hydrogen atom in the carboxamide group of acridine-4-carboxamides is most likely involved in a water-bridging hydrogen bond to the phosphate group in the DNA backbone ( 27 ). (ii) The absence of the basic moiety within the side chain of the secondary amides ( 9 ) or presence of carboxylic acid ( 17 ) led to complete loss of ability to increase melting temperature of the duplex as well. (iii) Compound 1 (no carboxamide moiety) also stabilised the ODN duplex slightly.…”

“…Preparation of 9-[(6-azidohexyl)amino]-N-butylacridine-4-carboxamide ( 9 ). This compound was prepared using the General procedure above but without the final conversion to hydrochloride.…”

Bifunctional Diblock DNA-Mediated Synthesis of Nanoflower-Shaped Photothermal Nanozymes for a Highly Sensitive Colorimetric Assay of Cancer Cells

“…Statistically, the mortality rate of cancers has ranked second after wars. , Therefore, early diagnosis of cancers appears to be particularly important for achieving a relatively high survival rate . Until now, various sensing methods, including but not limited to electrochemical, − optical, , magnetic, pressural, fluorescent, − and colorimetric ones, − served as excellent alternatives for cancer cell analysis. Among them, the colorimetric method has attracted much attention due to its visualization ability, low cost, and usage of simple instrumentation …”