A New Method For Sensitive Detection Of Telomerase Activity Without PCR Amplification

Telomeres are essential nucleoprotein structures that define the terminal segments of linear chromosomes in eukaryotes, and telomeres are essential for chromosome integrality and cell division. Because of DNA incomplete replication process, chromosome 3'ends progressively shorten during consecutive cell divisions, when lack sufficient telomeric repeats, the mechanism of apoptosis was activated. Human telomerase is a ribonucleoprotein reverse transcriptase that catalyzes the addition of the telomeric repeats onto the 3'-end of the human telomere chromosomes keeping telomeres extent, leading cell immortalization. Thus telomerase was selected as a universal tumor marker. The sensitive detection of telomerase activity could be crucially important for early cancer diagnosis and understanding the pathogenesis of disease. To date, a number of approaches to detect telomerase activity have been developed, some disadvantage such as sensitivity, assurance still exist. It is urgently needed to develop a high sensitive, rapid, reliable, cost effective strategy for telomerase activity detection. On the context, this paper puts forward a new strategy for telomerase activity detection aimed at developing simple, rapid, reliable, low-cost method of sensitive detection of telomerase activity.The main research contents and results are summarized as the following three sections.1. Fluorescent Detection of Telomerase Activity at Single-cell Level Based on Triple Amplification in homogeneous solutionHerein, we developed a PCR-free and label-free fluorescent strategy for facile, reliable and highly sensitive assay of human telomerase activity from crude cancer cell extracts. A G-quadruplex-selective fluorescent dye, N-methyl mesoporphyrin IX (NMM), was utilized as signal probe. Two hairpin probes with hidden G-quadruplex strand in their stem were designed as assembly components of strand displacement reaction (SDR). In this strategy, one telomerase reaction product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA) of SDR for achieving first step amplification. Then, strand displacement reaction led to the formation of G-quadruplex at the both end of two hairpin DNA probes for realizing second step amplification. Finally, the re-released T-DNA initiated another cycle of SDR, resulting in a significant increase in the fluorescence intensity of NMM. By taking advantage of triple signal amplification, the telomerase activity in the HeLa extracts equivalent to 1-3000 cells was detected in homogeneous solution. Telomerase activities of different cell lines, including cancer cells and normal cell, were also successfully evaluated. Meanwhile, the inhibition effect of 3'-azido-3'-deoxythymidine (AZT) was also investigated. Therefore, it offers a simple and reliable method for detecting telomerase activity at single-cell level without complex pre-modification of probe and enzyme auxiliary signal amplification, which has the merits of simplicity, rapid response, low cost and high reliability.2. Fluorescence Polarization Detection of Telomerase Activity at Single Cell Level Based on the Enhancement of Gold Nanoparticles Fluorescence polarization (FP) is a reliable, sensitive, and robust assay approach for studying biological interaction in homogeneous solution. To realize facile and reliable detection of telomerase activity, herein, for the first time, we developed a PCR-free fluorescent polarization strategy for sensitive detection of human telomerase activity at single cell level in homogenous solution based on gold nanoparticle (GNP) enhancement. Firstly, thiolated telomerase substrate (TS) primer is self-assembled onto the surface of GNP. In the presence of telomerase, TS primer was elongated to form a long single-stranded DNA containing numerous hexamer repeats (GGGTTA), which can hybridize with several short carboxyfluorescein (FAM)-modified complementary DNA (F-cDNA), leading to an increase in FP value due to the GNP enhancement and self-amplification of telomerase. So, the telomerase activity in the HeLa extracts equivalent to 1-1000 cells can be rapidly detected in homogeneous solution. Telomerase activities of different cell lines were also successfully evaluated. Meanwhile, the inhibition efficiency of telomerase inhibitor was studied, which holds great potential in screening telomerase-targeted anticancer drugs as well. Therefore, it offers a facile and reliable method for detecting telomerase activity at single-cell level, which has the merits of simplicity, rapid response, low cost, high sensitivity and reliability.3. Label-free Colorimetric Detection of Telomerase Activity via Triple AmplificationA label-free method is developed for colorimetry detecting telomerase activity using triple amplification mediated G-quadruplex-hemin DNAzymes. In this strategy, one telomerase reaction product contains several hexamer repeats which can hybridize with numerous assistant DNA to release a lot of trigger DNA (T-DNA). T-DNA can initiate cyclic strand displacement reaction between hairpin DNA H1 and H2. Through triple amplification, the simultaneous formation of two G-quadruplex at the both end of H1:H2 complex was occurred, the hemin-G-quadruplex DNAzyme can catalyze the H3O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to produce a colored product. Colorimetric functionality allows rapid preliminary discrimination of telomerase activity by the naked eye. Then the employment of UV-vis technique results in greatly improved detection sensitivity. Telomerase activity of 10 HeLa cells or above can be discriminated with the colorimetric functionality and UV-vis technique ensures that telomerase activity down to 1 HeLa cell can be sdetected. Telomerase-activities of different cell lines and telomerase inhibitor were also successfully investigated. With the ability of fast detection, outstanding sensitivity, and excellent selectivity, this strategy offers a convenient and specific method for telomerase activity detection, which exhibits great potential in the practical application in telomerase-based early stage cancer diagnosis.