Telomere Maintenance Mechanisms Define Clinical Outcome in High-Risk Neuroblastoma.

Neuroblastoma is a childhood cancer with heterogeneous clinical outcomes. To comprehensively assess the impact of telomere maintenance mechanism (TMM) on clinical outcomes in high-risk neuroblastoma, we integrated the C-circle assay [a marker for alternative lengthening of telomeres (ALT)], TERT mRNA expression by RNA-sequencing, whole-genome/exome sequencing, and clinical covariates in 134 neuroblastoma patient samples at diagnosis. In addition, we assessed TMM in neuroblastoma cell lines (n = 104) and patient-derived xenografts (n = 28). ALT was identified in 23.4% of high-risk neuroblastoma tumors and genomic alterations in ATRX were detected in 60% of ALT tumors; 40% of ALT tumors lacked genomic alterations in known ALT-associated genes. Patients with high-risk neuroblastoma were classified into three subgroups (TERT-high, ALT+, and TERT-low/non-ALT) based on presence of C-circles and TERT mRNA expression (above or below median TERT expression). Event-free survival was similar among TERT-high, ALT+, or TERT-low/non-ALT patients. However, overall survival (OS) for TERT-low/non-ALT patients was significantly higher relative to TERT-high or ALT patients (log-rank test; P < 0.01) independent of current clinical and molecular prognostic markers. Consistent with the observed higher OS in patients with TERT-low/non-ALT tumors, continuous shortening of telomeres and decreasing viability occurred in low TERT-expressing, non-ALT patient-derived high-risk neuroblastoma cell lines. These findings demonstrate that assaying TMM with TERT mRNA expression and C-circles provides precise stratification of high-risk neuroblastoma into three subgroups with substantially different OS: a previously undescribed TERT-low/non-ALT cohort with superior OS (even after relapse) and two cohorts of patients with poor survival that have distinct molecular therapeutic targets. SIGNIFICANCE: These findings assess telomere maintenance mechanisms with TERT mRNA and the ALT DNA biomarker C-circles to stratify neuroblastoma into three groups, with distinct overall survival independent of currently used clinical risk classifiers.

A facile DNA/RNA nanoflower for sensitive imaging of telomerase RNA in living cells based on "zipper lock-and-key" strategy.

The sensitive imaging of telomerase RNA (TR) in living cells is crucial for improved guidance in cancer clinical diagnosis because its expression level is closely related to malignant diseases. The efficient delivery of multiple nucleic acid probes to target cells is critical for nucleic acid-based methods to successfully image low-abundance TR in living cells. While novel nanomaterials enhance delivery efficiency, uncontrolled loading and slow intracellular release remain major challenges for multiple-probe delivery. Here, we designed a facile DNA/RNA nanoflower (NF) to perform the controlled loading of multiple probes and rapid intracellular release based on the "zipper lock-and-key" strategy. First, a long RNA generated by rolling circle transcription acts as both the "smart zipper lock" and the delivery carrier to alternately lock multiple functional DNAs through DNA-RNA base pairing, and the resulting RNA/DNA hybrids self-assemble into packed NFs. The functional DNAs include the fluorescence molecular beacon H1 for TR recognition, H2 for hybrid chain reaction (HCR) and DNA-cholesterol for size control. After NF internalization by the cells, the intracellular RNase H acts as the "key" to specifically open the DNA/RNA NFs by cleaving the RNA in the DNA/RNA hybrid, releasing high amounts of H1 and H2 in a confined space and thereby facilitating the HCR amplification analysis of cytoplasmic TR. With the addition of a DNA-nuclear localization peptide component in the same NF, nuclear TR can also be sensitively detected. Compared with the regular H1/H2 mixture, the DNA/RNA NFs produced a higher-contrast fluorescence signal. This indicated that the proposed strategy allowed the side arms of H1/H2 to be sealed into the RNA sequence-programmed "zipper lock" by controlled loading, avoiding mutual nonspecific H1/H2 hybridization. In addition, due to the fast kinetics of the RNase endonuclease reaction, the loaded H1/H2 was quickly released. Furthermore, the strategy was successfully used to assay the expression levels of TR in HeLa, HepG2 and HL-7702 cells, demonstrating that this approach holds the potential for the sensitive detection of low-abundance biomarkers in living cells.

PAQMAN: Protein-nucleic acid affinity quantification by MAss spectrometry in nuclear extracts.

In recent years, various mass spectrometry-based approaches have been developed to determine global protein-DNA binding specificities using DNA affinity purifications from crude nuclear extracts. However, these assays are semi-quantitative and do not provide information about interaction affinities. We recently developed a technology that we call Protein-nucleic acid Affinity Quantification by MAss spectrometry in Nuclear extracts or PAQMAN, that can be used to determine apparent affinities between multiple nuclear proteins and a nucleic acid sequence of interest in one experiment. In PAQMAN, a series of affinity purifications with increasing bait concentrations and fixed amounts of crude nuclear extracts are combined with isobaric stable isotope labeling and quantitative mass spectrometry to generate Hill-like Kd curves for dozens of proteins in a single experiment. Here, we apply PAQMAN to determine apparent affinities for a genetic variant, rs36115365-C, which regulates TERT expression and is associated with an increased risk to develop various malignancies. Furthermore, we describe a detailed protocol for this method including important quality checks.

Telomerase-triggered DNAzyme spiders for exponential amplified assay of cancer cells.

A highly flexible electrochemical assay based on target-triggered DNAzyme spiders was proposed for the detection of telomerase. The DNAzyme-telomerase substrate primers (D-TSP) containing Cu2+-dependent DNAzymes serve as recognition elements, and primers of telomerase. Telomerase extracted from Hela cells recognize the D-TSP and elongated with DNA sequence repeats. A synthetic telomerase product hybridized with scaffold sequences of two DNAzyme-tethered probes on the basis of the mechanism of the proximity-ligation assay. The three-leg DNAzyme spiders has been assembled and initiated the autonomous hybridization/nicking/displacement cycles on substrate modified surface. The cleaved ferrocene-labeled fragements are adsorbed on gold surface leading to an increase in the electrochemical signal. As a result, the one input target, telomerase, release large amount of ferrocene-labeled DNA strands, achieving an exponential signal amplification and an excellent improvement in sensitivity over single molecule or two-component 'sandwich' binding complexes. Our proposed biosensor showed a nonlinear dependence with Hela cell numbers, ranging from 25 to 2000 with a detection limit of 10 cells. Telomerase activities from different cell lines were also successfully evaluated. Our electrochemical strategy based on target-triggered DNAzyme spiders was enzyme-free, PCR-free, simple in operation which indicated that it expected to expand the scope of DNA nanotechnology in the areas of clinical diagnosis.

Highly sensitive fluorescence biosensor for intracellular telomerase detection based on a single patchy gold/carbon nanosphere via the combination of nanoflare and hybridization chain reaction.

How to in situ detect intracellular telomerase activity with high sensitivity still faces many challenges. This paper constructs a new fluorescence biosensing platform for the sensitive detection of intracellular telomerase activity via the combination of nanoflare and hybridization chain reaction (HCR)-based signal amplification on a single patchy gold/carbon nanosphere (PG/CNS), which has two or more distinct parts and allows hybridized-DNA (HS-DNA/Primer-DNA/Flare-DNA) and H1/H2-DNA (a pair of cross complementary DNA hairpins) to bind onto their surfaces via Au-S bond and electrostatic interaction, respectively. In the presence of telomerase, Primer-DNA (telomerase primer) extends at its 3' end to produce a telomeric repeated sequence, resulting in the release of Flare-DNA followed by the recovery of the fluorescence. Subsequently, the released Flare-DNA further initiates cross hybridization of H1 and H2 DNA from mimic-HCR system to amplify the fluorescence signal. The in vivo confocal microscopy studies demonstrate that resulting sensor can enter into the cancer cells such as A549 cells, and lead to the increase in luminescence, which is stronger than the sensor without the HCR-based signal amplification system. A linear relationship between the fluorescence intensity and the amount of A549 cells is observed, and the limit of detection of the sensor reaches about 280 A549 cells.

Strand displacement amplification-coupled dynamic light scattering method to detect urinary telomerase for non-invasive detection of bladder cancer.

Despite huge successes achieved by strand displacement amplification (SDA) and gold nanoparticles (AuNPs) in biomolecules sensing, the strategy of combination of SDA and AuNPs-based dynamic light scattering (DLS) for a biomolecule sensing is unexplored. Here we developed a non-invasive, SDA-based DLS method for the diagnosis of bladder cancer by detecting telomerase activity in human urine. In the presence of telomerase, the telomerase substrate (TS) primer was elongated with repeating sequences of (TTAGGG)n, and the resulting product triggers SDA between the hairpin deoxyribonucleic acid (DNA) and the Primer. The SDA product can be recognized by the oligonucleotide-modified AuNPs probes, resulting in DLS measurable AuNPs aggregation. The assay displayed a detection limit of 3 MCF-7 cells with a signal-to-noise ratio of 3 in a dynamic range of 5-1000 cells. The method was simple, reliable and has been successfully applied in the detection of telomerase in urine with good accuracy, selectivity and reproducibility. Moreover, only urine samples from bladder cancer patients induced a significant change in the average hydrodynamic diameter, indicating practical applicability of the method for the non-invasive diagnosis of bladder cancer.

A label-free electrochemical biosensor for ultra-sensitively detecting telomerase activity based on the enhanced catalytic currents of acetaminophen catalyzed by Au nanorods.

An electrochemical biosensor was designed for the determination of telomerase activity using an enzyme-free, PCR-free, and convenient electrochemical strategy. In this work, the electrochemical biosensor was constructed through the functionalization of Au nanorods with a carboxylic group (AuNRs-3) and subsequent immobilization with capture DNA (cDNA) for sensing telomerase activity. Upon telomerase triggered extension, the telomerase activity is related to the amount of the adsorbed electrocatalyst, leading to the different electrochemical signals for readout. Integrating with the efficient electrocatalysis of AuNRs-3-cDNA towards oxidation of acetaminophen, the prepared biosensor exhibits a wide dynamic correlation of telomerase activity from 1 × 102 to 1.04 × 107 HeLa cells mL-1 with a sensitivity of 2.68 HeLa cell mL-1 and the limit of detection was calculated to be 52.81 HeLa cells mL-1 under the optimal experimental conditions. Furthermore, the application of this electrochemical biosensor would provide the great potential for analysis of telomerase activity, revealing a powerful platform for early diagnosis of cancers.

Simultaneous detection of telomerase and miRNA with graphene oxide-based fluorescent aptasensor in living cells and tissue samples.

Telomerase and microRNAs (miRNAs) as important biomarkers are closely related to cancers. Simultaneous detection of telomerase activity and miRNAs would be beneficial to improve the specificity and reliability. Here, we establish a telomerase and miRNA-21 (miR-21) simultaneous sensing platform with graphene oxide-based fluorescent aptasensors (GOFA) including graphene oxide (GO), template strand (TS) primer and fluorophore-labeled telomerase/miR-21 oligonucleotides. Owing to π-π stacking interaction, TS primer and telomerase/miR-21 probes would be loaded onto GO, resulting in fluorescence quenching. However, in the presence of the telomerase or miR-21, the double-stranded oligonucleotides would be away from the GO surface attribute to the hybridization between the extended TS primers and telomerase probe as well as miR-21 and miR-21 probe, leading to obvious fluorescence recovery. We found that GOFA could simultaneously detect telomerase activity and miR-21 with low background signal, high sensitivity and simplified operation. Moreover, GOFA could be used for accurately detecting telomerase activity and miRNA in living cells and cancer patient tissue sample. This sensing platform shows great potential in improving the accuracy in clinical diagnosis of cancer.

Modular ssDNA binding and inhibition of telomerase activity by designer PPR proteins.

DNA is typically found as a double helix, however it must be separated into single strands during all phases of DNA metabolism; including transcription, replication, recombination and repair. Although recent breakthroughs have enabled the design of modular RNA- and double-stranded DNA-binding proteins, there are currently no tools available to manipulate single-stranded DNA (ssDNA). Here we show that artificial pentatricopeptide repeat (PPR) proteins can be programmed for sequence-specific ssDNA binding. Interactions occur using the same code and specificity as for RNA binding. We solve the structures of DNA-bound and apo proteins revealing the basis for ssDNA binding and how hydrogen bond rearrangements enable the PPR structure to envelope its ssDNA target. Finally, we show that engineered PPRs can be designed to bind telomeric ssDNA and can block telomerase activity. The modular mode of ssDNA binding by PPR proteins provides tools to target ssDNA and to understand its importance in cells.

Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

Quantitative assays for measuring human telomerase activity and DNA binding properties.

Telomerase is the ribonucleoprotein enzyme that catalyzes the processive addition of the telomeric DNA repeat 5'-TTAGGG-3' onto chromosome ends. In addition to its fascinating biochemical and enzymatic properties, clinical interest in telomerase stems from its dysregulated expression in ∼90% of human cancers, representing a broad spectrum of diseases. Exploiting telomerase as a therapeutic target and hence identifying and/or evaluating potential inhibitors requires quantitative measurement of its activity. This article presents procedures for measuring multiple aspects of telomerase enzymology that are relevant to both fundamental biochemistry and drug discovery: direct activity assays, DNA binding affinity, DNA dissociation, and cell-based over-expression of the active enzyme complex.

Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo.

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.

Measuring Telomerase Activity in Senescent Human T Cells Upon Genetic Modification.

Telomerase, a RNA-dependent DNA polymerase that adds telomeric DNA at the 3' ends of eukaryotic chromosomes, is essential for the lifelong preservation of the proliferative potential of antigen specific T lymphocytes. However, senescent T cells that have low telomerase activity, short telomeres and lack of replicative capacity accumulate in old humans, patients with chronic viral infections and cancer. The mechanisms inhibiting telomerase in these cells are poorly understood. Here I describe a strategy that was successfully applied to identify pathways causing telomerase dysfunction in primary human senescent T lymphocytes. Such strategy couples lentiviral vector-based gene manipulations to functional and signaling readouts directly ex vivo, in humans.

Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system.

Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting.

Design and development of PCR-free highly sensitive electrochemical assay for detection of telomerase activity using Nano-based (liposomal) signal amplification platform.

Telomerase, which has been detected in almost all kinds of cancer tissues, is considered as an important tumor marker for early cancer diagnostics. In the present study, an electrochemical method based on liposomal signal amplification platform is proposed for simple, PCR-free, and highly sensitive detection of human telomerase activity, extracted from A549 cells. In this strategy, telomerase reaction products, which immobilized on streptavidin-coated microplate, hybridized with biotinylated capture probes. Then, dopamine-loaded biotinylated liposomes are attached through streptavidin to biotinylated capture probes. Finally, liposomes are ruptured by methanol and the released-dopamine is subsequently measured using differential pulse voltammetry technique by multi-walled carbon nanotubes modified glassy carbon electrode. Using this strategy, the telomerase activity extracted from 10 cultured cancer cells could be detected. Therefore, this approach affords high sensitivity for telomerase activity detection and it can be regarded as an alternative to telomeric repeat amplification protocol assay, having the advantages of simplicity and less assay time.

A PCR-free fluorescence strategy for detecting telomerase activity via double amplification strategy.

As a universal tumor biomarker, research on the activity and inhibition of telomerase is of great importance for cancer diagnosis and therapy. Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its application has been significantly limited by amplification related errors and time-consuming procedure. To address the limitations of PCR-based protocol, a dual amplification fluorescence assay was developed for PCR-free detecting telomerase activity. Briefly, we designed an arch-structure DNA probe to specifically control strand displacement reaction and subsequent enzyme-aided amplification. Telomerase substrate (TS) primer was extended by telomerase to form long elongation products which contain several TTAGGG repeat units. So, one elongation product can release more than one trigger DNA (t-DNA) via strand displacement reaction to realize first amplification. Subsequently, t-DNA specifically opened molecular beacon (MB) to restore the fluorescence of MB. Meanwhile, t-DNA was recycled by the aid of nicking endonuclease to continuously open more and more MBs, leading to a second amplification. Owing to the double amplification strategy, the proposed method allowed the measurement of telomerase activity in crude cell extracts equivalent to 5 HeLa cells and 10 CCRF-CEM cells without PCR amplification. Besides, the influence of telomere-binding ligands on the telomerase activity demonstrated that the proposed method holds the potential to evaluate the inhibition efficiency of telomerase inhibitors.

Analysis of telomerase activity based on a spired DNA tetrahedron TS primer.

The development of sensitive telomerase biosensors is hindered by the restricted accessibility of telomere strand (TS) primer and the limited enzyme reaction space, which is mainly confined by the vertical distance. In this work, we designed an electrochemical telomerase biosensor based on a spired DNA tetrahedron TS primer (STTS). By adding a rigid dsDNA spire onto the top of the DNA tetrahedron, we successfully regulated the distance between the TS primer and the surface, and thus greatly facilitated the telomerase elongation on surface. The signal-to-noise ratio was 2 times higher than TSP without the spire structure. The limit of detection was calculated to be lower than 10 HeLa cells, which is at least 2 magnitudes lower than other surface extension-based electrochemical telomerase sensors without amplification. The practicability of STTS sensor was also demonstrated by analysing various other cell lines including cancer cells, stem cells of high telomerase activity and somatic cells of low telomerase activity.

Single gold nanoplasmonic sensor for clinical cancer diagnosis based on specific interaction between nucleic acids and protein.

Plasmonic nanomaterials reveal noble optical properties for next-generation biosensors. Nanoplasmonic biosensors have become simple, sensitive, smart, and consistent with advanced healthcare programs requirements. Notably, an individual nanoparticle analysis can yield unique target information, based on which the next-generation biosensor is revolutionary for end-point detection (single or multiplex), and can be functionally extended to biological phenomena monitoring. Here, we present a single nanoplasmonic sensing technology based on localized surface plasmon resonance for label-free and real-time detection of highly reliable cancer markers (mutant gene and telomerase) in clinical samples. The sensor specifically detects mutant DNA, and can detect telomerase from as few as 10 HeLa cells. This approach can be easily translated to detect other pathological targets with high sensitivity and specificity, and monitor key interactions between biomolecules such as nucleic acids and proteins during disease development in real time. This system has great potential to be further developed for on-chip and simultaneous analysis of multiple targets and interactions.

The telomerase reverse transcriptase subunit from the dimorphic fungus Ustilago maydis.

In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

Inventory of telomerase components in human cells reveals multiple subpopulations of hTR and hTERT.

Telomerase is the ribonucleoprotein (RNP) enzyme that elongates telomeric DNA to compensate for the attrition occurring during each cycle of DNA replication. Knowing the levels of telomerase in continuously dividing cells is important for understanding how much telomerase is required for cell immortality. In this study, we measured the endogenous levels of the human telomerase RNP and its two key components, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). We estimate ∼ 240 telomerase monomers per cell for HEK 293T and HeLa, a number similar to that of telomeres in late S phase. The subunits were in excess of RNPs (e.g. ∼ 1150 hTR and ∼ 500 hTERT molecules per HeLa cell), suggesting the existence of unassembled components. This hypothesis was tested by overexpressing individual subunits, which increased total telomerase activity as measured by the direct enzyme assay. Thus, there are subpopulations of both hTR and hTERT not assembled into telomerase but capable of being recruited. We also determined the specific activity of endogenous telomerase and of overexpressed super-telomerase both to be ∼ 60 nt incorporated per telomerase per minute, with Km(dGTP) ∼ 17 µM, indicating super-telomerase is as catalytically active as endogenous telomerase and is thus a good model for biochemical studies.