Monitoring Of Telomerase Activity Research Articles

An Enzyme-Free Cyclic Amplification Strategy for Intracellular Telomerase Activity Detection

Sensitive detection of telomerase and its inhibitor are of great significance to early diagnosis and treatment of malignant tumors. A cascade signal amplification strategy based on cyclic strand displacement reaction was designed to detect telomerase activity in cell lysates and living tumor cells. The primer can be elongated with repeated sequences of (TTAGGG) <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">n</sub> due to the presence of telomerase. The telomerase extension product (TEP) competes C-DNA with G-DNA by forming more stable duplex. Thus, G-DNA was released and trigger the cyclic strand displacement reaction. Two molecular beacons (MB1 and MB2) were applied to fulfill the cyclic amplification reaction and report the fluorescence signal. Compared with unamplified OMB strategy, the limit of detection for telomerase activity with amplified TMB strategy was enhanced by two orders of magnitude. It provides a new enzyme-free and PCR-free amplification method for quantification and monitoring of telomerase activity in living tumor cells. It can also be utilized for distinguishing tumor cells from normal cells by in situ imaging. The monitoring of telomerase activity with inhibitor demonstrated its potential applications in the discovery of anti-cancer drugs.

Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers.

Methods for detecting and quantifying disease biomarkers in biofluids with high specificity and sensitivity play a pivotal role in enabling clinical diagnostics, including point-of-care tests. The most widely used molecular biomarkers include proteins, nucleic acids, hormones, metabolites, and other small molecules. While numerous methods have been developed for analyzing biomarkers, most techniques are challenging to implement for clinical use due to insufficient analytical performance, high cost, and/or other practical shortcomings. For instance, the detection of cell-free nucleic acid (cfNA) biomarkers by digital PCR and next-generation sequencing (NGS) requires time-consuming nucleic acid extraction steps, often introduces enzymatic amplification bias, and can be costly when high specificity is required. While several amplification-free methods for detecting cfNAs have been reported, these techniques generally suffer from low specificity and sensitivity. Meanwhile, the quantification of protein biomarkers is generally performed using immunoassays such as enzyme-linked immunosorbent assay (ELISA); the analytical performance of these methods is often limited by the availability of antibodies with high affinity and specificity as well as the significant nonspecific binding of antibodies to assay surfaces. To address the drawbacks of existing biomarker detection methods and establish a universal diagnostics platform capable of detecting different types of analytes, we have developed an amplification-free approach, named single-molecule recognition through equilibrium Poisson sampling (SiMREPS), for the detection of diverse biomarkers with arbitrarily high specificity and single-molecule sensitivity. SiMREPS utilizes the transient, reversible binding of fluorescent detection probes to immobilized target molecules to generate kinetic fingerprints that are detected by single-molecule fluorescence microscopy. The analysis of these kinetic fingerprints enables nearly perfect discrimination between specific binding to target molecules and any nonspecific binding. Early proof-of-concept studies demonstrated the in vitro detection of miRNAs with a limit of detection (LOD) of approximately 1 fM and >500-fold selectivity for single-nucleotide polymorphisms. The SiMREPS approach was subsequently expanded to the detection of rare mutant DNA alleles from biofluids at mutant allele fractions of as low as 1 in 1 million, corresponding to a specificity of >99.99999%. Recently, SiMREPS was generalized to protein quantification using dynamically binding antibody probes, permitting LODs in the low-femtomolar to attomolar range. Finally, SiMREPS has been demonstrated to be suitable for the in situ detection of miRNAs in cultured cells, the quantification of small-molecule toxins and drugs, and the monitoring of telomerase activity at the single-molecule level. In this Account, we discuss the principles of SiMREPS for the highly specific and sensitive detection of molecular analytes, including considerations for assay design. We discuss the generality of SiMREPS for the detection of very disparate analytes and provide an overview of data processing methods, including the expansion of the dynamic range using super-resolution analysis and the improvement of performance using deep learning algorithms. Finally, we describe current challenges, opportunities, and future directions for the SiMREPS approach.

Potentiometric Characterization of Telomerase Activity Using a Copper(ii)-pyrophosphate Complex with a Copper ion-selective Electrode

A novel potentiometric sensing platform was designed for real-time electronic monitoring of telomerase activity using a copper(II) ion-selective electrode (Cu-ISE). The target-induced coordination of pyrophosphate ion with copper ion (Cu2+) was utilized for the detection of the enzyme activity. Upon telomerase introduction, a DNA primer was elongated in the presence of deoxyribonucleoside triphosphates, accompanying formation of the by-product (pyrophosphate ion; P2O74-). The generated pyrophosphate ions chemically coordinated with free copper ions to form the copper(II)-pyrophosphate complex, thereby resulting in a decrease in the number of free copper ions present in solution. With the addition of telomerase, the electrode potential on the Cu-ISE increased relative to the background signal. The results indicated that the copper(II)-pyrophosphate system exhibited good potentiometric response for the detection of telomerase activity, and allowed the determination of the analyte in HeLa cell extract at concentrations as low as 36 cells mL−1. Moreover, the Cu-ISE-based sensing platform afforded good reproducibility, thus representing a useful approach for practical use in quantitative telomerase activity assays.

Highly sensitive fluorescence quantification of intracellular telomerase activity by repeat G-rich DNA enhanced silver nanoclusters.

As an important biomarker for early cancer diagnosis and a valuable therapeutic target, the detection and monitoring of telomerase activity has attracted extensive attention. Herein, we constructed a novel fluorescent reporter to detect the intracellular telomerase activity based on guanine enhanced sliver nanoclusters (Ag NCs). In this system, a telomerase substrate (Ts) was designed to be extended by telomerase to give the repeat TTAGGG sequence as G-rich DNA, which was complementary to the nine base sequence to form a self-hairpin structure. The proximity of G-rich DNA to the as-prepared Ag NCs resulted in a 13-fold enhancement of fluorescence intensity due to the electron transfer from guanine to the Ag NCs. Therefore, the fluorescent probe could realize the detection of telomerase activity. Furthermore, the probe was successfully used to distinguish normal cells from cancer cells and to monitor the real time telomerase activity response upon treatment with an inhibiting model drug. This detection technique is simple, with excellent biocompatibility, inexpensive and expands the applications of the Ag NC probe.

Cardiotoxicity in rabbits after long-term nandrolone decanoate administration.

Abuse of anabolic androgenic steroids is linked to a variety of cardiovascular complications. The aim of our study was to investigate the possible cardiovascular effects of nandrolone decanoate on young rabbits using echocardiography, histology and monitoring of telomerase activity, oxidative stress and biochemical markers. Fourteen rabbits were divided into three administration groups and the control group. Doses of 4mg/kg and 10mg/kg of nandrolone decanoate, given intramuscularly and subcutaneously, two days per week for six months were applied. A 4-months wash-out period followed. Focal fibrosis and inflammatory infiltrations of cardiac tissue were observed in the high dose groups. Thiobarbituric acid-reactive species (TBARS) levels were significantly increased in the high dose groups, while catalase activity decreased. Myocardial Performance Index (MPI) is the main echocardiographic index primarily affected by nandrolone administration in rabbits. Despite the preserved systolic performance, histological lesions observed associated with distorted MPI values, point to diastolic impairment of the thickened myocardium due to nandrolone treatment. Oxidative stress accumulates and telomerase activity in cardiac tissue rises. Subcutaneous administration seems to be more deleterious to the cardiovascular system, as oxidative stress, telomerase activity and biochemical markers do not appear to return into normal values in the wash-out period.

New method to measure telomerase activity by transcription-mediated amplification and hybridization protection assay.

Telomerase is a ribonucleoprotein complex that uses RNA as a template for the addition of telomeric repeats. The development of the telomeric repeat amplification protocol (TRAP), a sensitive PCR-based assay, has facilitated the detection of telomerase activity in small tissue and tumor samples. Telomerase activity is expected to be a new diagnostic and prognostic marker of human cancer. In this study, we applied a non-PCR-based transcription-mediated amplification (TMA) and hybridization protection assay (HPA) to the measurement of telomerase activity by modification of both primers in TMA. We demonstrated that the modified TMA can detect and measure telomerase activity. TMA/HPA is as sensitive and reproducible as conventional TRAP, but is both faster and easier to perform. Furthermore, we found that TMA/HPA was influenced minimally by TRAP inhibitors that may come from clinical samples. TMA/HPA, which is easy, rapid, and applicable to a high-throughput format, should be clinically useful for the detection and monitoring of telomerase activity.