Sensing telomerase: From in vitro detection to in vivo imaging

We reviewed recent advances in telomerase assays including both in vitro detection and in vivo imaging.


Introduction
At the end of a chromosome, there is a region of repetitive nucleotide sequences (TTAGGG for human cells) called the telomere. 1 During natural cell division, the telomere length may be progressively shortened due to chromosome replication, leading to cellular aging and senescence. 2 This shortened telomere may be replenished by telomerase. 3 Telomerase is a ribonucleoprotein reverse transcriptase which consists of two molecules: the catalytic subunit of telomerase reverse transcriptase (TERT) and the TERT template of telomerase RNA (TERC). 4,5 TERT employs TERC to add the repeating sequence to the 3 0 end of a chromosome, preventing the shortening of the natural telomere. 6 Notably, the Nobel Prize in Physiology/ Medicine (2009) was awarded to Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak for their discovery of how chromosomes are protected by telomeres and the enzyme telomerase. [7][8][9] Recent research has demonstrated that the activation of telomerase activity may cause cellular immortality and cancers, 10 while the inactivation of the telomerase may accelerate cell aging. 11 The telomerase activity is repressed in most somatic human cells, while an elevated telomerase level is observed in over 85% of cancer cells, 12 including gastric cancer, 13 colorectal cancer, 14 cervical cancer, 15 pancreatic cancer, 16 breast cancer, 17 urothelial cancer, 18,19 and high-risk neuroblastoma. 20 Therefore, the telomerase may function as a universal biomarker for cancer diagnosis. Weinberg and colleagues found that the inhibition of telomerase by a mutant catalytic subunit of human telomerase may lead to the death of tumor cells, 21 22 indicates that telomerase may serve as an attractive target for anti-cancer therapies and anti-telomerase drug development. [23][24][25] For example, a telomerase template antagonist, GRN163, can inhibit telomerase activity and cause the suppression of tumor growth. 26 As a result, the accurate quan-tication of the telomerase is of great importance to clinic diagnosis and cancer therapy.
Recently, great efforts have been put into the development of efficient methods for telomerase assay since its discovery in 1985. 27 The most classical method is the polymerase chain reaction (PCR)-based telomeric repeat amplication protocols (TRAPs), 28 in which telomerase products are amplied and measured by PCR. The TRAP methods, however, are susceptible to the cell extract-induced inhibition. Alternatively, a series of new telomerase assays have emerged in recent years with the progress in bioanalytical chemistry and material sciences. Since the publication of the only review about the telomerase assay in 2012, 29 great advance has been made in this area, especially in the in vivo telomerase assay using novel nanomaterials. In this Minireview, we focus on the development of novel telomerase assays in the last ve years (2012-2016). These assays may be divided into two categories: in vitro telomerase quantication; and in vivo telomerase imaging. We introduce the assay principle and compare their performance. Meanwhile, we discuss the current challenges and future directions as well.

In vitro telomerase assays
In the in vitro telomerase assays, the target enzyme is extracted from the cells, and then quantied by various detection approaches including uorescent, colorimetric, electrochemiluminescent, Raman scattering spectroscopy, and chemiluminescent methods.

Fluorescent assay
The uorescent method is a widely used strategy for telomerase assay. The telomerase products may be directly/indirectly monitored by uorescent labels, such as zinc(II)-protoporphyrin IX (ZnPPIX), SYBR Green (SG), molecular beacon, quantum dot (QD) and Cy5. The uorescent methods may be divided into three categories: (1) amplication-free assay; (2) amplicationassisted assay; and (3) single QD-based assay.
2.1.1 Amplication-free uorescent assay. The telomerase is able to generate short tandem repeats of TTAGGG, which can form G-quadruplex oligomers. The integration of ZnPPIX into G-quadruplex may induce the increase of ZnPPIX uorescence by 9-fold. 30 Willner demonstrated a label-free uorescent method for telomerase assay using ZnPPIX (Fig. 1). 30 In the presence of dNTPs, telomerase extends the primer, producing multiple G-quadruplex sequences which subsequently bind ZnPPIX to generate an enhanced uorescent signal. This assay is very simple without the involvement of any amplication steps, and it may measure telomerase activity originating from 380 AE 20 cells per mL. In addition, this method is versatile and may be applied for the detection of DNA and adenosine-5 0triphosphate (ATP) using a specic hairpin probe and aptamer. Bo Tang obtained his PhD degree at Nankai University in 1994. Since then, he has been working as a professor at Shandong Normal University. His research focuses on analytical chemistry and nanotechnology. 2.1.2 Amplication-assisted uorescent assay. To improve the detection sensitivity, amplication approaches are introduced into the telomerase assays. Weizmann demonstrated the use of exponential isothermal amplication of the telomere repeat (EXPIATR) for real-time uorescent detection of telomerase activity (Fig. 2). 31 Unlike PCR amplication in TRAP-based telomerase, 28 the isothermal amplication does not need precise temperature control and sophisticated instruments. 32 This assay involves two primers (i.e., the nicking telomerase substrate primer and the nicking uorescent reporter probe primer) and three enzymes (i.e., DNA polymerase, nicking endonuclease and telomerase), with two primers containing the recognition sequence of the nicking endonuclease, respectively. In the presence of telomerase, the nicking telomerase substrate (NTS) may generate extension products which may function as the template for DNA amplication, initiating an exponential isothermal amplication reaction in the presence of DNA polymerase, nicking endonuclease and two primers. As a result, a number of double-stranded DNA (dsDNA) products are obtained. Telomerase activity can be real-time detected by the nicking uorescent reporter probe (NFRP) which can monitor the amplication process ( Fig. 2A) and SYBR Green I which can stain the amplication products of dsDNAs specically (Fig. 2B). This assay is very simple, without the involvement of thermal cycling, and it can detect telomerase activity in a single HeLa cell within 25 min.
Zhang demonstrated the use of telomerase-triggered isothermal exponential amplication for highly sensitive detection of telomerase activity. 33 In this assay, the extension products of the telomerase are amplied by an exponential amplication reaction, generating multiple 8-17 DNAzyme sequences which may be detected by molecular beacons in the presence of cofactor Pb 2+ . 34 This assay can measure telomerase activity at the single-cell level, and may be further applied for the screening of telomerase inhibitors.
2.1.3 Single QD-based assay. Due to their high quantum yield, good stability against photobleaching, narrow luminescence bands and size-tunable luminescence spectra, semiconductor quantum dots (QDs) have been widely used for the detection of various biomolecules. 35 Especially, the integration of uorescence resonance energy transfer (FRET) with QDs enables the homogeneous detection of DNA, microRNA and proteins. 36 Zhang developed a single QD-based biosensor for the sensitive detection of telomerase activity (Fig. 3). 37 In the presence of telomerase, the primer is extended by telomerase and simultaneously labelled by Cy5 with Cy5-dATP as the fuel. The Cy5-labeled extension products hybridize with the biotinylated capture probes and subsequently assemble on the surface of QD via biotin-streptavidin interaction to form the Cy5-dsDNA-QD assembly, leading to efficient FRET from the QD donor to the Cy5 acceptor. The telomerase activity may be simply quantied by counting the Cy5 signals at the single-molecule level. This assay is very sensitive, with a detection limit of 7 cells per mL, which is improved by 9-fold compared to the ensemble uorescence spectrum measurement. This assay can be further applied for the screening of anti-cancer drugs.

Colorimetric assay
The colorimetric method possesses the advantages of simplicity and being cost-effective, and its signal can be directly monitored by the naked eye. Qu demonstrated the use of primer-modied gold nanoparticles (AuNPs) for visualization detection of telomerase activity (Fig. 4). 38 In the absence of telomerase, the primer-modied AuNPs aggregate in a dened salt concentration, leading to the change in color. Meanwhile, in the presence of telomerase, the extension of the primer by telomerase prevents the AuNPs from aggregation, and no color change is observed. This assay enables  a simple and fast quantication of telomerase with a detection limit as low as 1 HeLa cell per mL. It can be further used to screen telomerase inhibitors for the discovery of anticancer agents.
In addition, based on the idea that L-cysteine may stimulate the aggregation of AuNPs, Willner developed an AuNP-based colorimetric method for telomerase detection. 39 In this assay, the enzyme products of telomerase may fold into G-quadruplex structures in the presence of K + ions and hemin, which possesses horseradish peroxidase mimicking functions and may catalyze the oxidation of L-cysteine into cysteine. As a result, the aggregation of AuNPs is prevented, and a distinct color change is observed. The assay can sensitively detect telomerase from 293T cancer cell extracts with a detection limit of 27 cells per mL, providing a potential point-of-care sensing platform for cancer diagnosis.

Electrochemical assay
The electrochemiluminescent method enables label-free and sensitive detection of biomolecules with a low background signal. 40 Qu demonstrated the development of an electrochemiluminescent biosensor for the sensitive detection of telomerase activity (Fig. 5). 41 They introduced the meso-tetra-(4-N,N,N-trimethylanilinium) porphyrin (TAPP) which had positively charged groups to prevent the aggregation of graphene. Meanwhile, they employed Ru(bpy) 3 2+ as the signal reporter and Tween 20 as the blocking agent to prevent the nonspecic binding of proteins in cell extract. The glassy carbon electrode was modied by TAPP-functionalized chemically converted graphemes (CCG). The negatively charged phosphate backbone and nucleotide base in the telomerase primer made it absorb on the grapheme surface through electrostatic attraction and p-p stacking. Subsequently, the negatively charged DNA brings the positively charged Ru(bpy) 3 2+ to the surface of the glassy carbon electrode through electrostatic attraction, generating an ECL signal. The elongation of the primer by telomerase produces a longer oligonucleotide which attracts more Ru(bpy) 3 2+ to the electrode surface, resulting in an enhanced ECL signal. This assay can sensitively measure telomerase activity with a detection limit of as low as 10 HeLa cells per mL. Recently, a series of electrochemical methods have been reported for telomerase assay using Methylene Blue 42 and hexaammineruthenium(III) chloride 43 as the reporters. In addition, metalorganic frameworks have been used in the development of electrochemical methods for telomerase assays. 44,45

Raman scattering spectroscopy-based assay
Surface-enhanced Raman scattering (SERS) may provide spectral ngerprint signatures of a specic target without interference from non-specic molecules. 46 Zong demonstrated the use of SERS for sensitive detection of telomerase activity (Fig. 6). 47 They prepared reporting gold nanoparticles (AuNPs) and capturing gold (Au) shell-coated magnetic nanobeads (MBs), respectively. The reporting AuNPs are modied with the Raman reporter 5,5 0 -dithiobis (2-nitrobenzoic acid) (DTNB) and a telomeric repeat complementary oligonucleotide (ATE); the capturing Au shell-coated MBs are modied with   a telomerase-substrate oligonucleotide (TS primer). In the presence of telomerase, the TS primers are elongated, generating tandem telomeric repeats. The hybridization of telomeric repeats with ATEs leads to the formation of AuNP-DTNB-MB complexes. Aer magnetic separation, a distinct SERS signal can be detected. Meanwhile, in the absence of telomerase, no telomeric repeat is produced, and no SERS signal is observed due to the absence of the AuNP-DTNB-MB complex. This assay is very sensitive and even the telomerase from 1 tumor cell per mL can be detected.

Chemiluminescent assay
Zhang demonstrated the use of two-stage isothermal amplication-mediated chemiluminescence for the ultrasensitive detection of telomerase activity (Fig. 7). 48 In the presence of telomerase, the substrate primer is extended, producing telomere repeats of (TTAGGG) n , which may function as the templates for strand displacement amplication (SDA). With the addition of primer, polymerase and nicking enzyme, multiple catalytic DNAzyme sequences and telomere repeats of (TTAGGG) n are generated. The resultant telomere repeats may subsequently function as the primers to trigger an isothermal exponential amplication reaction (EXPAR) and generate numerous catalytic DNAzyme sequences. These catalytic DNAzyme sequences can bind hemin to form G-quadruplex nanostructures which may catalyze the generation of luminolmediated chemiluminescence signals. Meanwhile, in the absence of telomerase, no amplication reaction is initiated, and no chemiluminescence signal is observed. This assay is highly sensitive, and it can detect the telomerase activity from a single HeLa cell without the involvement of any labeled DNA probes.
3 In vivo imaging of intracellular telomerase activity In addition to the above in vitro telomerase assays, a series of novel imaging methods for in vivo detection of telomerase activity have been developed recently. These imaging methods enable real-time tracking of dynamic telomerase processes in living cells, beneting the study of its physical role in disease development and drug response. Lou demonstrated the use of positively charged TPE-Py molecules for the imaging of telomerase in living cells (Fig. 8). 49 The TPE-Py is a kind of aggregation-induced emission (AIE) dye with weak uorescence in the separation state but intense uorescence in the aggregation state. 50 The telomerase substrate is labelled with a quencher. In the absence of telomerase, the uorescence originating from the binding of TPE-Py to the probe is quenched efficiently as a result of FRET from TPE-Py to the quencher. Aer being transferred into living cells, the extension of the substrate by telomerase generates a long DNA sequence with repeats of TTAGGG, which can bind TPE-Py molecules to generate a strong uorescence signal. This assay possesses signicant advantages of high stability and superior photostability, holding great potential in clinical diagnosis and telomerase-related drug screening.
The introduction of novel nanomaterials such as mesoporous silica nanoparticle (MSN) signicantly improves the in vivo assay performance. The MSN has the distinct characteristics of unique pore structure, biocompatibility and ease of functionalization, and is suitable for intracellular research. 51 Ju demonstrated the use of a telomerase-responsive mesoporous MSN probe for the uorescent imaging of intracellular telomerase activity (Fig. 9). 52 They prepared MSN and the wrapping DNA (O1), respectively. The MSN contains uorescein in the mesopores and black hole uorescence quencher (BHQ) on the inner walls of the mesopores. The O1 consists of the telomeric repeats and the telomerase substrate. In the absence of telomerase, the MSN probe is sealed by the O1, and uorescein is quenched by BHQ. In the presence of telomerase, the extension of substrate by telomerase leads to the removal of the O1 from  the MSN surface and consequently the release of uorescein which can be imaged by confocal microscopy. This assay enables switchable and real-time tracking of telomerase activity in living cells, and it can be used to monitor the change of intracellular telomerase activity in response to drugs.
The above method 52 involves complicated probe preparation, high background signal and multiple reaction processes. To simplify the procedures, Ju further demonstrated the use of gold nanoparticles and the nicked molecular beacon for onestep imaging of intracellular telomerase activity. 53,54 They designed a nicked molecular beacon with a nick at the 5 0 -end stem which separates the beacon into two parts: the telomerase primer sequence and a loop structure (Fig. 10). 53 The loop structure is labeled with Cy5 at 5 0 -end and modied by thiol at the 3 0 -end. The MB is conjugated to an AuNP via the thiol-labeled 3 0 -end, and the Cy5 is quenched by AuNP via FRET between Cy5 and AuNP. In the presence of telomerase, the extension of the primer by telomerase leads to the opening of hairpin and consequently the recovery of Cy5 uorescence as a result of the separation of Cy5 from the AuNP surface. The Cy5 uorescence lights up the telomerase. This assay exhibits good specicity and high sensitivity, and it may be applied for the discrimination of tumor cells from normal cells. In addition, Ding demonstrated the integration of nucleic acid-based signal amplication with a molecular beacon for a intracellular telomerase assay. 55 In addition to the uorescent imaging methods, 49,52,53 Kuang demonstrated the use of in situ Raman scattering spectroscopy for the intracellular telomerase assay (Fig. 11). 56 They designed a Cy5-tagged reporter probe (RS) and four single-stranded DNAs (ssDNAs, S1, S2, S3, and S4) which contain a complementary sequence to the telomerase primer (TP). The TP and RS may hybridize with the specic sequences of the four ssDNAs, leading to the formation of gold nanoparticle pyramids and consequently the generation of a high Raman signal. In the presence of telomerase, the extension of TP by telomerase releases RS from the pyramid scaffold, resulting in a decrease of the Raman signal. This assay may be used for in situ monitoring of intracellular telomerase activity in cell extracts with a linear range from 1 Â 10 À14 to 5 Â 10 À11 IU and a detection limit of as low as 6.2 Â 10 À15 IU. Meanwhile, through monitoring the uorescence signal of the Py-Cy5 nanostructure by confocal microscopy, the intracellular telomerase can also be uorescently imaged in living cells with a detection limit of 9.6 Â 10 À15 IU. Moreover, this assay shows signicant advantages of

Summary and outlook
Accurate quantication of telomerase activity plays a crucial role in clinical diagnosis and anti-cancer drug development, and great progress has been made in this area. In this Minireview, we summarize the recent advance in telomerase assays, including both in vitro assay and intracellular imaging. The in vitro telomerase activity may be monitored by a variety of approaches such as uorescent, 30,31,33,37 colorimetric, 38 electrochemiluminescent, 41 Raman scattering spectroscopy, 47 and chemiluminescent assays. 48 These in vitro assays possess the advantages of simplicity, good selectivity and high sensitivity. They cannot, however, be applied for in vivo/in situ monitoring of intracellular telomerase activity. The in vivo imaging of telomerase may provide direct information about its role in cancer progression and its response to drug treatment. With the introduction of novel nanomaterials, the in vivo imaging of telomerase in living cells may be achieved. 49,52,53,56 Both in vitro and in vivo telomerase assays make a great contribution to biomedical research and clinical diagnostics.
Notably, it still remains a great challenge to develop an ideal telomerase assay that satises the demands of rapidity, simplicity to operate, low cost, high sensitivity, good selectivity, and high-throughput at the same time. In addition, the in vivo monitoring of telomerase is still in its initial stage, and great efforts should be put into improving its performance in a complex cell environment. With the discovery of novel nanomaterials and the introduction of both new uorescent labels and efficient labelling strategies, we believe that the development of in vitro/in vivo telomerase assays may greatly facilitate clinical diagnosis and drug screening in the near future.