Target-controlled in situ formation of G-quadruplex DNAzyme for a sensitive visual assay of telomerase activity.

It is of great importance to achieve facile and reliable detection of telomerase because it is an important cancer biomarker. The complex components of cell extracts and ultra-low concentration of telomerase makes it more difficult to realize a simple and sensitive visual detection of telomerase activity. Herein, a facile and sensitive visual strategy was developed for the detection of telomerase based on the telomerase-controlled in situ formation of a G-quadruplex-hemin DNAzyme. To avoid the influence of the complex components of cell extracts, a telomerase substrate (TS) was immobilized onto the surface of magnetic beads (MBs) to form a MB/TS complex. MB/TS incubated with telomerase can add several TTAGGG repeat units to the 3' terminal of TS. After magnetic separation and washing, these G-rich elongated DNA folded into numerous G-quadruplex-hemin DNAzymes under the aid of K+ and hemin, which efficiently catalysed the TMB/H2O2 reaction. Magnetic separation basically eliminated the non-specific background interference from other cell extracts and redundant hemin. Taking full advantage of the in situ formation of multiple catalysts, the telomerase activity could be sensitively evaluated by a color change of the TMB/H2O2 solution. The telomerase activity down to 1 HeLa cell per µL and 0.5 HeLa cell per µL can be measured by the naked eye and UV-vis spectroscopy, respectively. Due to the magnetic separation and enrichment, the sensitivity was obviously improved compared with the previous colorimetric assay. Meanwhile, the telomerase activity of 5 HeLa cells per µL in human serum can be visually detected. Therefore, this study provides a facile, cost-effective and robust colorimetric assay for the visual detection of telomerase activity, which holds great potential in telomerase-based cancer clinical diagnostics.

An ultra-sensitive colorimetric assay for reliable visual detection of telomerase activity.

The visual detection of a disease biomarker is a promising strategy due to its simplicity and low cost, but the complexity of biological samples limits its application. Herein, a reliable and ultrasensitive colorimetric assay was proposed for detecting telomerase activity in crude cancer cell extracts. A telomerase substrate (TS) primer was immobilized onto magnetic beads (MBs) to form a MB/TS complex. In the presence of telomerase and deoxyribonucleotide triphosphates (dNTPs), the TS primer was elongated by adding multiple telomeric repeats (TTAGGG)n to the 3' end of the TS. The telomeric repeats of the elongated TS (ETS) hybridized with its short complementary DNA (cDNA) to specifically capture peroxidase onto MBs. After magnetic separation, the activity of telomerase was detected by monitoring the change in the color or absorbance of the peroxidase-catalyzed H2O2/TMB reaction. Magnetic separation greatly eliminated the non-specific interference to ensure reliability and improve the signal-to-noise (S/N) ratio. The mean telomerase activity equivalent to 5 HeLa cells and 1 HeLa cell was reliably detected with the naked eye and UV-vis spectroscopy, respectively. More importantly, the telomerase activity of 5 and 20 HeLa cells was detected via UV-vis spectroscopy and the naked eye, respectively. The differences in the telomerase activity of four carcinoma cell lines and one normal cell line were discriminated visually. Even more strikingly, the telomerase activity of 10 and 50 HeLa cells in human serum was detected by change in the absorbance and color of the TMB/H2O2 solution, respectively. Therefore, it offers an ultrasensitive and reliable colorimetric assay for the visual detection of telomerase activity, which holds promising potential to detect telomerase activity in a complex pathological sample.

Point-of-Care Assay of Telomerase Activity at Single-Cell Level via Gas Pressure Readout.

Detection of telomerase activity at the single-cell level is one of the central challenges in cancer diagnostics and therapy. Herein, we describe a facile and reliable point-of-care testing (POCT) strategy for detection of telomerase activity via a portable pressure meter. Telomerase primer (TS) was immobilized onto the surface of magnetic beads (MBs), and then was elongated to a long single-stranded DNA by telomerase. The elongated (TTAGGG)n repeat unit hybridized with several short PtNP-functionalized complementary DNA (PtNPs-cDNA), which specifically enriched PtNPs onto the surfaces of magnetic beads (MBs), which were separated using a magnet. Then, nanoparticle-catalyzed gas-generation reaction converted telomerase activity into significant change in gas pressure. Because of the self-amplification of telomerase and enrichment by magnetic separation, the diluted telomerase equivalent to a single HeLa cell was facilely detected. More importantly, the telomerase in the lysate of 1 HeLa cell can be reliably detected by monitoring change in gas pressure, indicating that it is feasible and possible to study differences between individual cells. The difference in relative activity between different kinds of cancer cells was easily and sensitively studied. Study of inhibition of telomerase activity demonstrated that our method has great potential in screening of telomerase-targeted antitumor drugs as well as in clinical diagnosis.

High-throughput identification of telomere-binding ligands based on the fluorescence regulation of DNA-copper nanoparticles.

Formation of the G-quadruplex in the human telomeric DNA is an effective way to inhibit telomerase activity. Therefore, screening ligands of G-quadruplex has potential applications in the treatment of cancer by inhibit telomerase activity. Although several techniques have been explored for screening of telomeric G-quadruplexes ligands, high-throughput screening method for fast screening telomere-binding ligands from the large compound library is still urgently needed. Herein, a label-free fluorescence strategy has been proposed for high-throughput screening telomere-binding ligands by using DNA-copper nanoparticles (DNA-CuNPs) as a signal probe. In the absence of ligands, human telomeric DNA (GDNA) hybridized with its complementary DNA (cDNA) to form double stranded DNA (dsDNA) which can act as an efficient template for the formation of DNA-CuNPs, leading to the high fluorescence of DNA-CuNPs. In the presence of ligands, GDNA folded into G-quadruplex. Single-strdanded cDNA does not support the formation of DNA-CuNP, resulting in low fluorescence of DNA-CuNPs. Therefore, telomere-binding ligands can be high-throughput screened by monitoring the change in the fluorescence of DNA-CuNPs. Thirteen traditional chinese medicines were screened. Circular dichroism (CD) measurements demonstrated that the selected ligands could induce single-stranded telomeric DNA to form G-quadruplex. The telomere repeat amplification protocol (TRAP) assay demonstrated that the selected ligands can effectively inhibit telomerase activity. Therefore, it offers a cost-effective, label-free and reliable high-throughput way to identify G-quadruplex ligands, which holds great potential in discovering telomerase-targeted anticancer drugs.

A cytometric assay for ultrasensitive and robust detection of human telomerase RNA based on toehold strand displacement.

Human telomerase RNA (hTR), works as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes, is overexpressed in tumor cells and its concentration has a positive correlation with telomerase activity. The lack of facile and reliable method for detection of hTR in complex matric limited its application for clinical diagnosis. To address the limitation, herein, we proposed a facile and reliable flow cytometric assay for sensitive and specific detection of hTR by combing magnetic enrichment with signal amplification of DNA toehold strand displacement reaction (TSDR). Two hairpin DNA probes of TSDR are ingeniously designed, including biotinylated hairpin DNA1 (H1) and carboxyfluorescein (FAM)-labeled hairpin DNA2 (F-H2). Firstly, H1 was immobilized on streptavidin-functionalized magnetic beads (STV-MBs) through biotin-avidin interaction. In the presence of hTR DNA, TSDR between H1 and F-H2 was triggered to continuously form H1/H2 duplex, resulting in a "turn on" fluorescence on the surface of MBs. Due to fluorescence amplification of TSDR and magnetic enrichment, hTR-DNA can be sensitively, specifically and facile analyzed by flow cytometry and fluorescence microscopy imaging. The detection limit of flow cytometry is 0.3pM, which is superior to those of most existing approaches. Moreover, the proposed strategy can be successfully utilized to detect hTR in complex biological media as well. Therefore, an enzyme-free amplification approach is provided for robust and rapid detecting hTR DNA, which offers a facile, reliable and sensitive method for studying disease-related gene.

Homogeneous and ultrasensitive detection of telomerase activity via gold nanorod-based fluorescence resonance energy transfer.

As a universal biomarker, telomerase is one of the promising targets for cancer diagnosis and therapy. Therefore, it is meaningful to develop facile, robust and sensitive methods for evaluation of telomerase activity. Herein, combined with fluorescence resonance energy transfer (FRET), we creatively designed a gold nanorod (GNR)-based FRET method to detect telomerase activity from cell extracts. As the signal probe, carboxyfluorescein-modified DNA probes (F-DNA) hold negative electricity. The electrostatic interaction between F-DNA and positively charged GNRs makes F-DNA close to GNRs, which leads to weak FRET between the F-DNA and GNRs. In the presence of telomerase, a telomerase substrate (TS) primer was elongated to form a long single-stranded DNA, which could hybridize with numerous F-DNA to form long dsDNAs. The strengthened electrostatic interaction leads to a more efficient FRET between GNRs and dsDNA. Therefore, the amplified fluorescence quenching efficiency can greatly improve the sensitivity. The telomerase activity in the HeLa extracts equivalent to 1 cell was detected sensitively without the polymerase chain reaction (PCR) amplification and enzyme auxiliary signal amplification. Moreover, this facile protocol can be used to distinguish tumor cells from normal cells and study the inhibition effect of telomerase inhibitors, which shows its potential application value in clinical diagnosis and drug screening.