Ratiometric Fluorescent Biosensor for Visual Discrimination of Cancer Cells with Different Telomerase Expression Levels.

Telomerase is inactive in normal somatic cells but highly activated in tumor cells to maintain their indefinite proliferation and immortal phenotype. As a specific marker for the generation and progress of almost all tumors, the detection of telomerase activity by classical PCR techniques has served in the biological research of tumors. However, the detection of in situ telomerase activity in cell extracts to evaluate the malignancy, progress, and metastasis of tumors remains a daunting challenge. Here, a precisely designed FRET-based ratiometric fluorescent oligonucleotide probe has achieved high-fidelity detection of telomerase activity for accurate discrimination of different cancer cells toward advanced diagnosis of tumors. Our method is superior to other methods in its capabilities to quantify telomerase activity in cell extracts and visualize various tumor cell extracts with different telomerase expression levels by the naked eye for clinical diagnosis. In particular, the ratiometric fluorescent probe used in the assay could exclude other experimental factors influence, and further avoid false positive signal generation. The method reported here could provide a reliable, accurate, and convenient way in medical diagnostics and therapeutic response assessment.

A dual-colored ratiometric-fluorescent oligonucleotide probe for the detection of human telomerase RNA in cell extracts.

Human telomerase RNA (hTR), which is one component of telomerase, was deemed to be a biomarker to monitor tumor cells due to its different expression levels in tumor cells and normal somatic cells. Thus far, plentiful fluorescent probes have been designed to investigate nucleic acids. However, most of them are limited since they are time-consuming, require professional operators and even result in false positive signals in the cellular environment. Herein, we report a dual-colored ratiometric-fluorescent oligonucleotide probe to achieve the reliable detection of human telomerase RNA in cell extracts. The probe is constructed using a dual-labeled fluorescent oligonucleotide hybridized with target-complemented Dabcyl-labeled oligonucleotide. In the presence of the target, the dual-labeled fluorescent oligonucleotide translates into a hairpin structure, which leads to the generation of the fluorescence resonance energy transfer (FRET) phenomenon under UV excitation. Compared to conventional methods, this strategy could effectively avoid false positive signals, and it not only possesses the advantages of simplicity and high specificity but also has the merits of signal stability and distinguishable color variation. Moreover, the quantitative assay of hTR would have a far-reaching impact on the telomerase mechanism and even tumor diagnosis research.