A Smart DNA Tweezer for Detection of Human ... - ACS Publications

Subscriber access provided by UNIV OF DURHAM

Article

A Smart DNA Tweezer for Detection of Human Telomerase Activity Xiaowen Xu, Lei Wang, Kan Li, Qihong Huang, and Wei Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05373 • Publication Date (Web): 15 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A Smart DNA Tweezer for Detection of Human Telomerase Activity Xiaowen Xu,† Lei Wang,‡ Kan Li,† Qihong Huang,§ and Wei Jiang*,† †

Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, School of Pharmaceutical Sciences, and §School of Life Sciences, Shandong University, Jinan 250100, China.

‡

ABSTRACT: Reliable and accurate detection of telomerase activity is crucial for better understanding its role in cancer cells and further exploring its function in cancer diagnosis and treatment. Here, we construct a smart DNA tweezer (DT) for detection of telomerase activity. The DT is assembled by three specially-designed single-stranded oligonucleotides: a central strand duallylabeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs’ complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG)n. TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toeholdmediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The detection limit of telomerase activity is equivalent to 141 cells/µL HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, TRPs owning 1, 2, 3, 4, and ≥ 5 telomeric repeats are identified to account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer.

Human telomerase is a ribonucleoprotein comprised of telomerase RNA and telomerase reverse transcriptase, catalyzing the addition of telomeric repeats (TTAGGG)n onto the 3’end of telomere.1 Telomerase is found to be over-expressed in most types of malignant cancer cells, which compensates the shortening of telomere during cell division and enables cancer cells to circumvent telomere-dependent pathway of cell mortality to divide infinitely.2 Telomerase inhibition can reduce its capability to elongate telomere and suppress continuous proliferation of cells that depends on telomere maintenance.3 Thus, telomerase has been regarded as a promising biomarker and a potential therapeutic target for cancer.4 Effective detection of telomerase is crucial for better understanding its role in cancer cells and further exploring its function in cancer diagnosis and treatment.5 Conventional methods for telomerase activity detection are direct primer extension assay6 and telomeric repeat amplification protocol (TRAP).7 To avoid polymerase-derived artifacts and radioactive/mutagenic hazards, emerging techniques including fluorescence,8-11 electrochemistry,12-15 colorimetry,16-19 chemiluminescence,20 surface plasmon resonance,21 surface enhanced Raman scattering22 have been developed. Although these methods achieve good sensitivity, most of them rely on single signal intensity for telomerase quantification, and are susceptible to internal factors of surrounding nuclease, protease and glutathione, as well as external factors of light source drifts and voltage fluctuations, which may generate degradation-induced false signals and compromises the reproducibility.23,24 Moreover, telomerase translocates along the newly synthesized sequence for the next telomeric repeat addition

and is a processive enzyme.25,26 It generates products of various lengths, and the length distribution is believed to reflect telomerase’s translocation efficiency that is necessary for catalysis mechanism study27 and telomerase’s subtle mutation that is important for disease diagnosis.28 Most of the abovementioned methods detect signals from a mixture of products, and do not identify the length distribution of products. Thus, new strategies for reliable and accurate detection of telomerase’s activity and product lengths are still urgently needed. Molecular machines drive molecules with controllable movements and perform special tasks when energy is added, showing appealing potential in the development of new sensors, intelligent devices and energy storage systems.29 DNA molecular machine is a kind of molecular machine made of assembled DNA structure that performs machinelike nanomechanical movement at micro- or nanoscopic scale.30 DNA molecular machine owns several unique advantages. Its building materials, DNA oligonucleotides, can be chemically synthesized, thermally renatured and readily modified, enabling DNA molecular machines facile to be constructed and characterized. Moreover, DNA sequences are highly programmable, and various DNA nanostructures can be assembled based on the specific base pairing,31 creating nano-objects with designer functions. A number of DNA molecular machines have been constructed, such as tweezer,32 walker,33 gear,34 metronome,35 transporter,36 and so forth. In addition, DNA is a powerful recognition element for a large scope of targets including nucleic acid fragments, metal ions, small molecules and proteins,37 endowing DNA molecular machine great potential in the development of sensors. Currently, most of DNA molecu-

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

lar machines are reported for cycle-based signal amplification in sensing application,38-41 and new functional DNA molecular machines still need to be explored to meet the broader detection requirements. Herein we construct a smart DNA tweezer (DT) for detection of telomerase activity and determination of product length distribution. The DT is assembled by three specially-designed single-stranded oligonucleotides: a central strand duallylabeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs’ complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG)n. TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toehold-mediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The ratio of fluorescence of acceptor to donor is employed for quantification. The detection limit of telomerase activity is equivalent to 141 cells/µL HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, the length distribution of TRPs is identified, with TRPs owning 1, 2, 3, 4, and ≥ 5 telomeric repeats accounting for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer. EXPERIMENTAL SECTION Materials. DNA oligonucleotides (Table S-1) and materials are detailed in the Supporting Information. Preparation of DT. Prior to DT preparation, the concentrations of oligonucleotides were quantified according to the UV absorbance at 260 nm and the molar extinction coefficients were calculated by sum of extinction coefficients of individual bases in each sequence.42 This process is very important since excess oligonucleotides weakens the effective binding of TRP to DT. Central strands DT-T1 and arm strands DT-T2, DT-T3 at the same concentration (1.5 µM for each) were annealed in 1 × NEB buffer 4 (20 mM Tris-Ac, 50 mM KAc, 10 mM Mg(Ac)2, 1 mM DTT, pH 7.9) by heating at 95 °C for 5 min and cooling slowly to room temperature, obtaining the stable DT. The DT was hold at 4 °C with a stock concentration of 0.5 µM for further use. For the verification of DT formation, 15% native polyacrylamide gel electrophoresis (PAGE) was performed in 1 × TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3) at a current of 30 mA, running 1.5 h at 15 °C. The gel was stained with 1 × SYBR Gold for 40 min, and photographed with GelDocTM XR+ imaging system (BioRAD Laboratories Inc., USA). Telomerase extension reaction. Telomerase was extracted from cells by the CHAPS method.43 Telomerase extension reaction was conducted by incubating 8.4 µL of 5 × TRAP buffer (100 mM Tris-HCl, 7.5 mM MgCl2, 315 mM KCl,

Page 2 of 18

0.025% Tween-20, 5 mM EGTA, pH 8.3), 4.2 µL of 10 mM dNTPs, 1 µL of 500 nM TS primer and 28.5 µL of cell extracts with different concentrations at 37 °C for 4 h. Then, telomerase was deactivated by heating at 90 °C for 10 min. For control experiments, telomerase extracts were heat-deactivated at 90 °C for 10 min, or pre-incubated with AZT (2 mM) at 37 °C for 12 hours before the extension reaction. For the TS elongation with purified telomerase enzyme, different concentrations of purified enzyme were added instead of cell extracts under otherwise identical conditions. Close and open operation of DT. 1 µL of 500 nM DT, 4.9 µL of 10 × NEB buffer 4 and 1.1 µL water was added into the above extension reaction mixture. The mixture was incubated at 37 °C for 3 h to strain DT to the closed state. After that, 1 µL of 500 nM RS was added and the mixture was incubated at 37 °C for another 4 h to relax DT to the open state. Overall, it took around 7.2 h to obtain a final result of TRPs concentration and around 11.2 h to obtain a final result of TRP length distribution. For DT operation with chemically synthetic telomeric product (CSTP), procedures were conducted as mentioned above except that the mixture of 8.4 µL of 5 × TRAP buffer, 4.2 µL of 10 mM dNTPs, 1 µL of 500 nM CSTP and 28.5 µL of CHAPS lysis buffer was substituted for the telomerase extension mixture. F-7000 spectrometer (Hitachi, Japan) was employed to measure fluorescence during the above processes. The excitation wavelength was 485 nm and the spectra were recorded from 510 nm to 650 nm with a scan speed of 240 nm/min. The excitation and emission slits were both set as 10 nm, and the voltage was set as 780 V. The reported errors for fluorescence data represent the mean and standard deviation from three independent measurements. RESULTS AND DISCUSSION Principle of DT for telomerase detection. The principle of DT for detection of telomerase activity and product length distribution is illustrated in Figure 1. DT is constructed by hybridization between central strand DT-T1 and arm strands DT-T2, DT-T3. It contains a 4-base single-stranded hinge acting as a flexible region for configuration change and two single-stranded overhangs (dark green and dark blue domains) acting as recognition sequences to TRP. DT structural change induced by TRP or RS and its resulting FRET variation constitute the basic detection principle. FRET is a quantum phenomenon occurring between two fluorophores.44 Excitation is transferred nonradiatively from a donor to an acceptor fluorophore through dipole-dipole interaction.45 As a result, the donor fluorescence is quenched, while the acceptor fluorescence becomes excited.46 It can be realized when the donor and acceptor molecules are within 1–10 nm of distance.47 The DT is initially in an open state, whose fluorescence donor (FAM, shown in green spark) and acceptor (TAMRA, shown in red dot) are far away from each other, maintaining a low FRET efficiency. First, under the action of telomerase, TS (light green domain) are elongated with numbers of telomeric repeats (light blue and light yellow domains), generating TRPs (Figure 1A). TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, leading to the approach of donor to acceptor and a high FRET efficiency (Figure 1B). The fluorescence ratio (FA/FD) is obtained by dividing acceptor emission intensity by donor emission intensity, correlating directly to the amount of TRP. Thus the close operation


Page 3 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Schematic illustration of the smart DNA tweezer for detection of telomerase activity and determination of product length distribution. The light blue and light yellow domains in TRP are both telomeric repeats. The light blue domain is the binding region for DT, while the light yellow domain is the toehold for the subsequent strand displacement.

of DT is utilized for detection of telomerase activity. Second, RS is added and hybridizes to TRP, first at the exposed toehold (light yellow domain) and then proceeding by branch migration. This results in the complete displacement of TRP from DT and the formation of a TRP-RS duplex. The closed tweezer returns to the open state and the FRET efficiency decreases (Figure 1C). The TRP length would be figured out by resolving the individual contribution that enables the reset of specific DT. Thus the open operation of DT is utilized for determination of TRP length distribution. Proof-of-concept study of DT close. We firstly verified the synthesis of DT and tested its hybridization with CSTP. As shown in Figure 2A, the bands in lane 1, lane 2 and lane 3 correspond to the central strand DT-T1 and arm strands DTT2, DT-T3, respectively. After annealing of those three strands, the bands of individual strand disappear while a new, strong band with retarded mobility is observed (lane 4), indicating the efficient synthesis of DT. To test the hybridization of DT, CSTP containing 3 telomeric repeats (CSTP-3R) that is characterized with a single band in lane 5, is complexed with DT. It is observed the formation of a strong band that migrates rather slowly (lane 6), as well as the diminishing of the CSTP3R band, suggesting the efficient hybridization of CSTP-3R to DT. The slower migrating rate of the DT in lane 6 than that in lane 4 results from that the hybridized CSTP-3R increases the molecular weight and negative charge of the original tweezer. To study the configuration change, fluorescence measurements were performed. The system containing DT and TS shows a strong fluorescence intensity of FAM donor at 520 nm and a low fluorescence intensity of TAMRA acceptor at 580 nm (Figure 2B), suggesting the donor and acceptor are separated and DT is in the open state with a low FRET efficiency. When CSTP is used instead of TS, the fluorescence intensity of FAM donor decreases while fluorescence intensity of TAMRA acceptor increases, suggesting the donor and acceptor get approached and DT is transformed to closed state with a high FRET efficiency. Emission of FAM is quenched by resonant energy transfer to TAMRA, leading to the

Figure 2. (A) Gel electrophoresis characterization of DT synthesis and its hybridization with CSTP. Lane M: marker, lane 1: DTT1 (500 nM), lane 2: DT-T2 (500 nM), lane 3: DT-T3 (500 nM), lane 4: DT (500 nM), lane 5: CSTP-3R (500 nM), lane 6: DT (500 nM) + CSTP-3R (500 nM). (B) Fluorescence spectra of DT (10 nM) complexed with TS (10 nM) and CSTP-3R (10 nM).

enhanced emission of TAMRA. We then tested DT close by TRP that is generated under the action of cervical cancer (HeLa) cellular extracted telomerase. In the absence of telomerase extracts, the system shows a low FRET efficiency, demonstrating that DT is initially open and the donor and acceptor are far away from each other (Figure 3A). However, in the presence of telomerase extracts, the system shows an obviously enhanced FRET efficiency, demonstrating that DT gets closed, with the donor and acceptor drawn close to each other. The TS elongation under the action of telomerase is confirmed by gel characterization of PCRamplified TRPs. A repeating pattern of elongated products is observed only in the presence of telomerase extracts (Figure 3B). The gel piece containing products was cut, and the DNA therein was recovered and sequenced. Continuous (TTAGGG)n sequences are found to be added on the 3’-end of the original TS (Figure S-1), validating the generation of TRPs under the action of telomerase extracts. The generated TRPs would then hybridize with the two overhangs of DT to strain it to closed state. Optimization of reaction conditions. We chose fluorescence ratio that refers to the ratio of the fluorescence of TAMRA acceptor to that of FAM donor for quantification. In comparison with the direct recording of FAM intensity, it records two emission intensities at different wavelengths, and analyzes the data based on FRET. The fluorescence ratio can be written as a function of FRET efficiency (E) to be FA/FD = E / (1-E) (deducted in Supporting Information). It is less subject to influence from cell extracts48 as well as photobleaching, the drifts of light sources and detectors,49 ensuring the detection reliability. Meanwhile, it obtains similar results across different detection platforms including spectrofluorometry, fluorescence microplate reader and confocal microscopy for the same reaction system, and can meet different detection requirements and is effective in data comparison.50 The reaction conditions were investigated using the value of fluorescence ratio change as a standard, which is formulated as eq.1. ∆(FA/FD) = (FA/FD)p-(FA/FD)a (1) In the formula, (FA/FD)p and (FA/FD)a represent the ratio of fluorescence intensity of acceptor to donor, under the



Page 4 of 18

Figure 4. (A) Fluorescence spectra of DT complexed with different concentrations of HeLa cells extracts. (B) Linear relationship between ∆(FA/FD) and the concentration of HeLa cells. Figure 3. (A) Fluorescence spectra of DT in the presence and absence of HeLa telomerase extracts (2850 cells/µL). (B) Gel electrophoresis characterization of TS elongation under the action of telomerase extracts. Lane 1: TS + HeLa extracts (2850 cells/µL), lane 2: TS - HeLa extracts, lane M: marker.

circumstances in the presence and absence of cell extracts, respectively. A big variation of ratio implies a dramatic configuration change of DT from open to closed state. The number of complementary bases between DT-T3 and TS primer plays an important role in the DT configuration change. On the one hand, insufficient complementary bases may increase the difficulty of closing DT by the short TRP that contains one telomeric repeat, weakening the positive response and influencing the subsequent study of TRP length distribution. On the other hand, excessive complementary bases may lead to a high background, since TS itself may close DT. As the number of complementary bases between DT-T3 and TS varies from 5 to 8, values of (FA/FD)p as well as (FA/FD)a both increase (Figure S-2A), suggesting that DT becomes more liable to be closed. The biggest value of ∆(FA/FD) is obtained with DT containing 6 complementary bases to TS (Figure S-2B), which is employed in the following study. Additionally, the concentration of DT used in the system was investigated. The value of ∆(FA/FD) gradually increases as the DT concentration varies from 2 nM to 10 nM, and then deceases as the DT concentration varies from 10 nM to 40 nM (Figure S-3). The reason should be that a low concentration of DT reduces its hybridization opportunity to TRP, while a high concentration of DT scales down the proportion of TRP-closed DT in the total amount. Thus 10 nM DT is chosen for the system. The time of telomerase extension reaction has a crucial effect on the generation of TRP and the subsequent DT close. It is manifested that the value of ∆(FA/FD) gradually increases and reaches a plateau at 4.0 h (Figure S-4), so 4.0 h of extension reaction time is used. To ensure a sufficient time for DT close, a series of closing reaction time were tested. The value of ∆(FA/FD) reaches a plateau until 3.0 h (Figure S-5), which is used as an optimal time for DT close. Analytical performance of DT for telomerase activity. Under the optimal conditions, the analytical performance of DT for telomerase activity was investigated. When the concentrations of HeLa cell extracts increase from 0 to 2850 cells/µL, the fluorescence intensity of FAM gradually decreases while fluorescence intensity of TAMRA gradually increases (Figure 4A). Along with the increasing concentration of HeLa cell extracts, the total amount of telomerase increases proportionally, resulting in a higher TRP level and a higher FRET

efficiency. A good linearity is obtained within HeLa cell concentration range from 280 cells/µL to 2850 cells/µL (Figure 4B). The linear function is ∆(FA/FD) = 9.037×10-5 × c 0.01277 (R2 = 0.994), where c is the concentration of HeLa cell extracts. The detection limit is estimated to be a telomerase activity equivalent to 141 cells/µL according to three times of standard deviation over the background. Although the sensitivity of the proposed method is lower than that of TRAP (Figure S-6), it is more suitable for quantification by measuring fluorescence, and does not involve PCR procedure thus can avoid PCR-related artifacts. The DT should belong to the “switch-off” sensor since the fluorescence of FAM decreases obviously while the fluorescence of TAMRA increases less obviously along with the increasing telomerase extracts. Its sensitivity is lower than some reported “switch-on” sensors, such as the single quantum dot-based biosensor51 and the fluorescent amphiphilic nucleic acid probes.52 This is caused by that the gain of “switch-off” sensor is limited since the target cannot suppress more than 100% of the original signal, but the gain of “switch-on” sensor can theoretically increase without limit.53 However, the “switch-off” sensor possesses its own advantages, including: 1) it can obtain better detection performance for the target whose binding affinity to DNA is low;54 2) it involves simpler designs and fewer steps thus is more convenient and cost-effective;55 3) it has the potential to be flexibly adapted with additional steps for the “switch-on” detection of more information of a target. The DT should also avoid the risk of false-negative signals that will cause ineffective cancer screening. The DNA nano-assembly shows resistance against degradation,56 thus DT is relatively stable for detection telomerase extracts, reducing the possibility of degradationinduced sensor invalidity and false-negative signals. Additionally, DT does not need any tool enzyme such as polymerase to accomplish the detection, and can be exempted from falsenegative signals caused by extracts-induced tool enzyme degradation. For detection of 300, 1500 and 2500 cells/µL, the relative standard deviations performed in three independent experiments are 3.1%, 1.9% and 1.9% within one day, and are 4.4%, 2.7% and 3.0% among three days, demonstrating a good reproducibility. Besides, DT also demonstrates similar response to purified telomerase enzyme (Figure S-7A), supporting that telomerase plays a role in the fluorescence change. The fluorescence ratio shows a linear relationship toward telomerase concentration from 6 × 10-9 IU/µL to 9 × 10-8 IU/µL (Figure S-7B). Through the standard addition of purified telomerase to cell extracts and measuring fluorescence of DT, it is calculated that 1.15 × 10-8 IU telomerase are present in 500 HeLa cells (Figure S-8). The result is in good accordance with


Page 5 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Specificity of DT for telomerase activity detection. The concentrations of all tested cells are 2850 cells/µL.

the previously measured telomerase content.57,58 To test the specificity of the strategy, DT is complexed with normal liver cell (HL-7702) extracts, heat-treated HeLa extracts, telomerase inhibitor-treated HeLa extracts and other types of cancer cell extracts at the concentration of 2850 cells/µL (Figure 5). As somatic cells, HL-7702 do not overexpress telomerase and their ∆(FA/FD) shows little difference from the background. The heat treatment can destroy reverse transcriptase protein and telomerase RNA,59 which deactivates telomerase and generates a ∆(FA/FD) value similar to the background. The telomerase inhibitor AZT strongly inhibits reverse transcriptase activities,60 and produces a ∆(FA/FD) value much lower than that of HeLa cell extracts. Other types of cancer cells, including liver cancer cells (HepG2), acute lymphoblastic leukemia cells (CEM), breast cancer cells (MCF-7), show high ∆(FA/FD) values, indicating the over-expression of telomerase activity in these cancer cells. The different ∆(FA/FD) values among these cancer cell extracts are attributed to unequal telomerase expression levels.61 It is revealed that telomerase activity is relatively high in HeLa cells while relatively low in HepG2 cells, which is consistent with the previous report.62 All those demonstrate that DT possesses good specificity to telomerase activity, and holds potential to differentiate active telomerase from inactive telomerase, as well as to differentiate cancer cell extracts from normal cell extracts. Proof-of-concept study of DT open. The DT open is studied prior to the determination of product length distribution. We employed toehold-mediated strand displacement63 to return the closed DT to open state, which requires an exposed toehold on DT. To resolve the contribution from each TRP with specific length, several DTs and RS are used. The DT bearing DT-T3 that contains complementary sequences to n of telomeric repeats is defined as DT-nR, and RS containing complementary sequences to n of telomeric repeats is defined as RS-nR. When DT-1R is complexed with HeLa cell extracts, it is observed a time-dependent fluorescence decrease of FAM and a time-dependent fluorescence increase of TAMRA (Figure 6A, 6B). This reveals that DT can get closed by aid of the one extended telomeric repeat. It also suggests that falsenegative signal caused by the inability of responding to TRPs containing a few telomeric repeats can be avoided. Further, when the RS-2R is introduced, it is observed a time-dependent fluorescence increase of FAM and a time-dependent fluorescence decrease of TAMRA (Figure 6C, 6D). This suggests a decrease of FRET efficiency and a successful open of DT by

Figure 6. Time-dependent fluorescence changes of FAM (A) and TAMRA (B) within DT-1R in the presence and absence of HeLa extracts (2850 cells/µL), and time-dependent fluorescence changes of FAM (C) and TAMRA (D) within DT-1R (10 nM) in the presence and absence of RS-2R (10 nM).

RS. In control experiments, the fluorescence of both FAM and TAMRA shows little change with time in the absence of cell extracts or RS-2R, confirming that the observed fluorescence change results from target-induced DT configuration change instead of photobleaching or structural laxity of closed tweezer. Parameter unification and rectification. DT-nR (n=1-4) with DT-T3 containing complementary sequences to 1-4 telomeric repeats, are used in the TRP length distribution measurement. Those DTs show almost similar fluorescence background (Figure S-9, black histogram), probably due to the flexibility of the single-stranded hinge connecting two arms. Furthermore, after complexed with telomerase extracts, the four DTs obtain similar FRET efficiency (Figure S-9, red histogram). This is because that the DT close has little relation with DT-T3 and TRP lengths since one telomeric repeat is sufficient (Figure 6A, B). Thereby, the values of FA/FD before and after interaction with telomerase for the four DTs can be unified. The open efficiency of DT by RS was then tested, with the purpose of rectifying DT open-induced fluorescence ratio change to reflect the actual closed DT. As shown in Figure S-10A, DT-1R gets closed upon its hybridization with CSTP-2R, inducing the enhanced fluorescence ratio. The closed DT-1R is then opened by RS-2R, and the open efficiency (OE) is calculated to be 92%, according to eq. 2. OE %

/ / / / / / / /

100% (2)

In the formula, (FA/FD)DT, (FA/FD)DT/CSTP and (FA/FD)DT/CSTP/RS represent the fluorescence ratio of acceptor to donor, for DT, DT/CSTP complex, and DT/CSTP/RS complex that plateau to the open or closed state of DT, respectively. With the same method, the open efficiencies of RS-(n+1)R to DT-nR/CSTP-(n+1)R complex (n = 2, 3, 4) are calculated to be 91%, 91% and 90%, respectively (Figure S-10B to S-10D). The four DTs’ open efficiencies are very close to each other and the mean value is 91%. In other words, RS opens up to 91%



Page 6 of 18

of the total closed tweezer, thus the fluorescence ratio is rectified to reflect the actual closed DT by TRP, according to eq. 3.

%

(3)

In the formula, (FA/FD)nR-M represents the measured fluorescence ratio of acceptor to donor for the DT-nR/RS-(n+1)R system, (FA/FD)nR represents the rectified ratio. Further test was conducted to verify that the fluorescence ratio correlates positively with the amount of TRPs. It is observed that as the concentration of CSTP-1R increases, the fluorescence of donor shows a gradual decrease while the fluorescence of acceptor shows a gradual increase (Figure S11A). Moreover, the concentration of CSTP-1R demonstrates a good linear relationship with ∆(FA/FD) (Figure S-11B). The linear function is ∆(FA/FD) = 0.1181 × c - 0.02577 (R2 = 0.992), where c is the concentration of CSTP-1R with an unit of nM. It suggests that the TRP amount can be compared through the fluorescence ratio. Determination of the TRP length distribution. According to the above results, DT-nR gets closed through hybridization with TRPs, and then gets reopen via the strand displacement by RS-(n+1)R. Only if the number of telomeric repeats in TRPs is no less than (n+1), TRP can be removed by RS(n+1)R from the DT-nR, resulting in the open of tweezer (Figure S-10D). The measurement of TRP length distribution is detailed in Figure 7. Under the action of telomerase, TS are elongated with various numbers of telomeric repeats (Figure 7A). According to the toehold-mediated strand displacement, only when the closed DT contains a protruding toehold (light orange domain), the TRP can hybridize with RS and get displaced to induce the DT open. We utilize DT-nR/RS-(n+1)R as each machine system, in which DT-nR gets closed by TRPs and gets reopen by RS-(n+1). To open DT-1R with RS-2R, TRPs should contain at least 2 telomeric repeats (Figure 7B). To open DT-2R with RS-3R, open DT-3R with RS-4R and open DT-4R with RS-5R, TRPs should contain at least 3, 4 and 5 telomeric repeats respectively (Figure 7C to 7E). As the flowchart shown in Figure 7F, through the combination of the individual machine systems into a circuit, one is able to determine the TRP length distribution. DT-nR/RS-(n+1)R (n=1-4) are complexed with TRPs respectively. Figure 8A shows that the values of FA/FD for DTnR decrease to different degrees upon the introduction of RS(n+1)R (n=1-4). For the system containing DT-nR and RS(n+1)R, the DT open only occurs when TRPs own ≥ (n+1) telomeric repeats, through the toehold of one telomeric repeatmediated strand displacement between RS and TRP. Therefore TRP with specific length can be calculated by subtracting the amount of TRPs that are longer than this specific length from the amount of total TRPs that enables the open of a given DT. The amount proportion (AP) of TRPs with specific numbers (SN) of telomeric repeats that account for the total amount of products is calculated according to following equations. F F , , FFAP %"#$%,',(,),* 100% 4 F F , , . FFIn Eq. 4, (FA/FD)nR represents the rectified fluorescence ratio of acceptor to donor for the DT-nR/RS-(n+1)R system. The

Figure 7. Elongation of TS with various numbers of telomeric repeats under the action of telomerase (A), the specific reaction of TRPs towards DT-1R/RS-2R (B), DT-2R/RS-3R (C), DT-3R/RS4R (D), DT-4R/RS-5R (E) systems, and a circuit to determine the TRP length distribution (F). The light yellow domain in TRP indicates the toehold for the subsequent strand displacement.

obtained AP means the amount proportion of TRPs that enable the DT-nR open. AP %"#0,',(,),* 1 AP %"#$%,',(,),*

1

1

100% 5

Eq. 5 is derived from Eq. 4, and the obtained AP means the amount proportion of TRPs that cannot open DT-nR. AP %"#','(,),* AP %"#0 AP %"#0 F F , , FF 100% 6 F F , , . FF-


Page 7 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. (A) Values of FA/FD for TRPs-closed DT-nR tweezer upon the introduction of RS-(n+1)R, and (B) amount proportion of TRPs with different numbers of repeats.

Eq. 6 is derived from Eq. 5, and the obtained AP means the amount proportion of TRP with specific number of telomeric repeats. Figure 8B shows the calculated proportion of TRPs with different numbers of telomeric repeats. Here we mainly focus on TRPs with 1 to 4 telomeric repeats because they are primarily considered in evaluation of telomerase mutation-related disease. The mutant of telomerase that is related with disease usually generates TRPs with less than 3 telomeric repeats.26 The telomeric repeat addition processivity calculated through TRPs of 1, 2, 3 and 4 telomeric repeats has been utilized as a standard to differentiate telomerase that is mutant in the telomerase reverse transcriptase.27 Additionally, there is a link between human disease such as dyskeratosis congenita and aplastic anaemia and mutations in human telomerase RNA, and their processivity difference from wild type is mainly manifested on TRPs with 1 to 4 telomeric repeats.28 Identifying the TRP length distribution can also provide a reference for the telomerase sensor design. It is revealed that TRPs bearing 1, 2, 3, 4, and ≥ 5 telomeric repeats account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The TRP length distribution is close to recently reported results by single molecule stochastic binding assay.64 However, DT provides a homogenous, direct way for TRP length distribution determination, which eliminates the confinement of TS onto heterogeneous interface that would reduce its accessibility to telomerase and complementary reporter DNA. For further comparison, direct primer elongation measurement is performed by labeling TS with radioactive 32P and manifesting products on the gel through autoradiograph. The rough amount proportion of each product is calculated according to band intensity. As shown in Figure S-12, TRPs bearing 1, 2, 3, 4, and ≥ 5 telomeric repeats account for 27.0%, 19.2%, 14.2%, 12.4%, and 27.2%, respectively. The result is consistent with the result obtained by DT, validating the credibility of analyzing TRP length distribution with the proposed DNA machine. For telling the size of TRPs, DT measures fluorescence instead of semi-quantitative band intensity thus is more accurate for quantification, and does not require radioactive/mutagenic hazards and particular experimental chamber. In comparison with traditional TRAP assay, it does not involve PCR thus avoids PCR-derived artifacts.65 Besides, TRAP assay requires counting and summing the bands intensity from all the TRPs to get a baseline reference, and certain TRPs may get neglected due to the limited sensitivity of gel imaging, causing the result deviation. Whereas DT obtains the baseline reference though simply measuring fluorescence of DT in the presence of TRPs, and is more convenient and accurate.

CONCLUSIONS In summary, we have demonstrated the detection of telomerase activity and product length distribution, based on a smart DT whose operation can be regulated. The DT close operation is utilized for telomerase activity detection, in which TRPs hybridize to the tweezer and strain the tweezer arms, inducing increased FRET efficiency. The DT open operation is utilized for product length distribution determination, in which RS displaces the TRPs via toehold-mediated strand displacement and relax the tweezer arms, inducing decreased FRET efficiency. The intact tweezer operation-derived fluorescence ratio avoids degradation-induced false-negative signals and minimizes ambient fluctuations. The DT shows good sensitivity and specificity toward telomerase extracts, and identifies the length distribution of TRPs. It will offer a new approach for reliable, accurate analysis of telomerase for better understanding its catalysis mechanism, role in cancer cells, and further exploring its function in cancer diagnosis and treatment. This work also demonstrates how to utilize the operation of a molecular machine for development of sensors in acquiring multiple information of a target, so as to better meet the practical assay requirements.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials, additional experimental details, formula derivation, DNA sequences (Table S-1), sequencing result of elongated products recovered from native PAGE gel (Figure S-1), values of ∆(FA/FD) versus number of complementary bases between DT-T3 and TS (Figure S-2), values of ∆(FA/FD) versus concentrations of DT (Figure S-3), values of ∆(FA/FD) versus telomerase extension reaction time (Figure S-4), values of ∆(FA/FD) versus closing reaction time of DT (Figure S-5), TRAP assay result for measuring HeLa cell telomerase extracts (Figure S-6), fluorescence response of DT towards purified telomerase enzyme (Figure S-7), calibration curve of standard addition method by DT for detection endogenous telomerase in HeLa cell extracts (Figure S-8), values of FA/FD for DT-nRs in the absence and presence of HeLa cell extracts (Figure S-9), toehold-mediated strand displacement reaction efficiency (Figure S-10), fluorescence spectra and linear relationship of DT complexed with CSTP (Figure S-11), evaluation of TRP length distribution by direct primer elongation measurement (Figure S-12).

AUTHOR INFORMATION Corresponding Author * Fax: +86 531 88564464. E-mail: [email protected].

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Nos. 21375078, 21475077, 21675100, 21675101 and 21705094), the China Postdoctoral Science Foundation (2015M582074), the Natural Science Foundation of Shandong Province (ZR2017BB032), the Postdoctoral Innovation Program Special Funds of Shandong Province (201603024) and the Fundamental Research Funds of Shandong University (11190075614003).



REFERENCES (1) Cohen, S. B.; Graham, M. E.; Lovrecz, G. O.; Bache, N.; Robinson, P. J.; Reddel, R. R. Science 2007, 315, 1850-1853. (2) Harley, C. B. Nat. Rev. Cancer 2008, 8, 167-179. (3) Cian, A. D.; Cristofari, G.; Reichenbach, P.; Lemos, E. D.; Monchaud, D.; Teulade-Fichou, M.-P.; Shin-ya, K.; Lacroix, L.; Lingner, J.; Mergny, J.-L. Proc. Natl. Acad. Sci. 2007, 104, 44, 17347-17352. (4) Wu, L.; Qu, X. Chem. Soc. Rev. 2015, 44, 2963-2997. (5) Wang, L.-J.; Ma, F.; Tang, B.; Zhang, C.-Y. Chem. Sci. 2017, 8, 2495-2502. (6) Morin, G. B. Cell 1989, 59, 521-529. (7) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L. C.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Science 1994, 266, 2011-2015. (8) Tian, L.; Weizmann, Y. J. Am. Chem. Soc. 2013, 135, 16611664. (9) Lou, X.; Zhuang, Y.; Zuo, X.; Jia, Y.; Hong, Y.; Min, X.; Zhang, Z.; Xu, X.; Liu, N.; Xia, F.; Tang, B. Z. Anal. Chem. 2015, 87, 6822-6827. (10) Hong, M.; Xu, L.; Xue, Q.; Li, L.; Tang, B. Anal. Chem. 2016, 88, 12177-12182. (11) Zhang, Z.; Jiao, Y.; Zhu, M.; Zhang, S. Anal. Chem. 2017, 89, 4320-4327. (12) Liu, X.; Li, W.; Hou, T.; Dong, S.; Yu, G.; Li, F. Anal. Chem. 2015, 87, 4030-4036. (13) Liu, X.; Wei, M.; Liu, Y.; Lv, B.; Wei, W.; Zhang, Y.; Liu, S. Anal. Chem. 2016, 88, 8107-8114. (14) Liu, Y.; Wei, M.; Liu, X.; Wei, W.; Zhao, H.; Zhang, Y.; Liu, S. Chem. Commun. 2016, 52, 1796-1799. (15) Ling, P.; Lei, J.; Ju, H. Anal. Chem. 2016, 88, 10680-10686. (16) Wang, J.; Wu, L.; Ren, J.; Qu, X. Small 2012, 8, 259-264. (17) Duan, R.; Wang, B.; Zhang, T.; Zhang, Z.; Xu, S.; Chen, Z.; Lou, X.; Xia, F. Anal. Chem. 2014, 86, 9781-9785. (18) Wang, J.; Wu, L.; Ren, J.; Qu, X. Nanoscale 2014, 6, 16611666. (19) Xu, X.; Wang, L.; Huang, Y.; Gao, W.; Li, K.; Jiang, W. Anal. Chem. 2016, 88, 9885-9889. (20) Wang, L.-J.; Zhang, Y.; Zhang, C.-Y. Anal. Chem. 2013, 85, 11509-11517. (21) Maesawa, C.; Inaba, T.; Sato, H.; Iijima, S.; Ishida, K.; Terashima, M.; Sato, R.; Suzuki, M.; Yashima, A.; Ogasawara, S.; Oikawa, H.; Sato, N.; Saito, K.; Masuda, T. Nucleic Acids Res. 2003, 31, e4. (22) Zong, S.; Wang, Z.; Chen, H.; Cui, Y. Small 2013, 9, 42154220. (23) Du, Y.; Lim, B. J.; Li, B.; Jiang, Y. S.; Sessler, J. L.; Ellington, A. D. Anal. Chem. 2014, 86, 8010-8016. (24) Yang, X.-J.; Zhang, K.; Zhang, T.-T.; Xu, J.-J.; Chen, H.-Y. Anal. Chem. 2017, 89, 4216-4222. (25) Greider, C. W. Mol. Cell. Biol. 1991, 11, 4572-4580. (26) Ren, X.; Li, H.; Clarke, R. W.; Alves, D. A.; Ying, L.; Klenerman, D.; Balasubramanian, S. J. Am. Chem. Soc. 2006, 128, 4992-5000. (27) Huard, S.; Moriarty, T. J.; Autexier, C. Nucleic Acids Res. 2003, 31, 4059-4070. (28) Chen, J.-L.; Greider, C. W. Embo J. 2003, 22, 304-314. (29) Leigh, D. A. Angew. Chem. Int. Ed. 2016, 55, 14506-14508. (30) Teller, C.; Willner, I. Curr. Opin. Biotechnol. 2010, 21, 376391. (31) Hong, F.; Zhang, F.; Liu, Y.; Yan, H. Chem. Rev. 2017, 117, 12584-12640. (32) Yurke, B.; Turberfield, A. J.; Mills Jr, A. P.; Simmel, F. C.; Neumann, J. L. Nature 2000, 406, 605-608. (33) Shin, J.-S.; Pierce, N. A. J. Am. Chem. Soc. 2004, 126, 1083410835. (34) Tian, Y.; Mao, C. J. Am. Chem. Soc. 2004, 126, 11410-11411. (35) Buranachai, C.; McKinney, S. A. Nano Lett. 2006, 6, 496-500.

Page 8 of 18

(36) Wang, Z.-G.; Elbaz, J.; Willner, I. Angew. Chem. Int. Ed. 2012, 51, 4322-4326. (37) Liu, J.; Cao, Z.; Lu, Y. Chem. Rev. 2009, 109, 1948-1998. (38) Li, D.; Wieckowska, A.; Willner, I. Angew. Chem. Int. Ed. 2008, 47, 3927-3931. (39) Wang, F.; Elbaz, J.; Willner, I. J. Am. Chem. Soc. 2012, 134, 5504-5507. (40) Zhang, H.; Lai, M.; Zuehlke, A.; Peng, H.; Li, X.-F.; Le, X. C. Angew. Chem. Int. Ed. 2015, 54, 14326-14330. (41) Xu, Z.; Liao, L.; Chai, Y.; Wang, H.; Yuan, R. Anal. Chem. 2017, 89, 8282-8287. (42) Xu, X.; Zhang, J.; Yang, F.; Yang, X. Chem. Commun. 2011, 47, 9435-9437. (43) Piatyszek, M. A.; Kim, N. W.; Weinrich, S. L.; Hiyama, K.; Hiyama, E.; Wright, W. E.; Shay, J. W. Methods Cell Sci. 1995, 17, 1-15. (44) Didenko, V. V. Biotechniques 2001, 31, 1106-1121. (45) Zhang, W.-H.; Ma, W.; Long, Y.-T. Anal. Chem. 2016, 88, 5131-5136. (46) Xie, N.; Huang, J.; Yang, X.; Yang, Y.; Quan, K.; Ou, M.; Fang, H.; Wang, K. ACS Sens. 2016, 1, 1445-1452. (47) Cao, Y.; Qian, R.-C.; Li, D.-W.; Long, Y.-T. Chem. Commun. 2015, 51, 17584-17587. (48) Shigeto, H.; Nakatsuka, K.; Ikeda, T.; Hirota, R.; Kuroda, A.; Funabashi, H. Anal. Chem. 2016, 88, 7894-7898. (49) Zhang, P.; Beck, T.; Tan, W. Angew. Chem. Int. Ed. 2001, 40, 402-405. (50) Chen, H. H.; Ho, Y.-P.; Jiang, X.; Mao, H.-Q.; Wang, T.-H.; Leong, K. W. Nano Today 2009, 4, 125-134. (51) Zhu, G.; Yang, K.; Zhang, C.-Y. Chem. Commun. 2015, 51, 6808-6811. (52) Jia, Y.; Gao, P.; Zhuang, Y.; Miao, M.; Lou, X.; Xia, F. Anal. Chem. 2016, 88, 6621-6626. (53) Xiao, Y.; Lubin, A. A.; Baker, B. R.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. 2006, 103, 16677-16680. (54) Li, N.; Ho, C.-M. J. Am. Chem. Soc. 2008, 130, 2380-2381. (55) Wang, R. E.; Zhang, Y.; Cai, J.; Cai, W.; Gao, T. Curr. Med. Chem. 2011, 18, 4175-4184. (56) Qian, H.; Tay, C. Y.; Setyawati, M. I.; Chia, S. L.; Lee, D. S.; Leong, D. T. Chem. Sci. 2017, 8, 1062-1067. (57) Qian, R.; Ding, L.; Yan, L.; Lin, M.; Ju, H. Anal. Chem. 2014, 86, 8642-8648. (58) Wang, W.; Huang, S.; Li, J.; Rui, K.; Bi, S.; Zhang, J.-R.; Zhu, J.-J. Chem. Sci. 2017, 8, 174-180. (59) Qian, R.; Ding, L.; Yan, L.; Lin, M.; Ju, H. J. Am. Chem. Soc. 2014, 136, 8205-8208. (60) Melana, S. M.; Holland, J. F.; Pogo, B. G. T. Clin. Cancer Res. 1998, 4, 693-696. (61) Wang, W.-J.; Li, J.-J.; Rui, K.; Gai, P.-P.; Zhang, J.-R.; Zhu, J.-J. Anal. Chem. 2015, 87, 3019-3026. (62) Wang, J.; Wu, L.; Ren, J.; Qu, X. Nanoscale 2014, 6, 16611666. (63) Zhang, D. Y.; Winfree, E. J. Am. Chem. Soc. 2009, 131, 17303-17314. (64) Su, X.; Li, Z.; Yan, X.; Wang, L.; Zhou, X.; Wei, L.; Xiao, L.; Yu, C. Anal. Chem. 2017, 89, 3576-3582. (65) Zhou, X.; Xing, D. Chem. Soc. Rev. 2012, 41, 4643-4656.


Page 9 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents artwork



Figure 1. Schematic illustration of the smart DNA tweezer for detection of telomerase activity and determination of product length distribution. The light blue and light yellow domains in TRP are both telomeric repeats. The light blue domain is the binding region for DT, while the light yellow domain is the toehold for the subsequent strand displacement. 83x57mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 10 of 18

Page 11 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (A) Gel electrophoresis characterization of DT synthesis and its hybridization with CSTP. Lane M: marker, lane 1: DT-T1 (500 nM), lane 2: DT-T2 (500 nM), lane 3: DT-T3 (500 nM), lane 4: DT (500 nM), lane 5: CSTP-3R (500 nM), lane 6: DT (500 nM) + CSTP-3R (500 nM). (B) Fluorescence spectra of DT (10 nM) complexed with TS (10 nM) and CSTP-3R (10 nM). 83x43mm (300 x 300 DPI)



Figure 3. (A) Fluorescence spectra of DT in the presence and absence of HeLa telomerase extracts (2850 cells/µL). (B) Gel electrophoresis characterization of TS elongation under the action of telomerase extracts. Lane 1: TS + HeLa extracts (2850 cells/µL), lane 2: TS - HeLa extracts, lane M: marker. 83x45mm (300 x 300 DPI)


Page 12 of 18

Page 13 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (A) Fluorescence spectra of DT complexed with different concentrations of HeLa cells extracts. (B) Linear relationship between ∆(FA/FD) and the concentration of HeLa cells. 83x33mm (300 x 300 DPI)



Figure 5. Specificity of DT for telomerase activity detection. The concentrations of all tested cells are 2850 cells/µL. 60x48mm (300 x 300 DPI)


Page 14 of 18

Page 15 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Time-dependent fluorescence changes of FAM (A) and TAMRA (B) within DT-1R in the presence and absence of HeLa extracts (2850 cells/µL), and time-dependent fluorescence changes of FAM (C) and TAMRA (D) within DT-1R (10 nM) in the presence and absence of RS-2R (10 nM). 83x67mm (300 x 300 DPI)



Figure 7. Elongation of TS with various numbers of telomeric repeats under the action of telomerase (A), the specific reaction of TRPs towards DT-1R/RS-2R (B), DT-2R/RS-3R (C), DT-3R/RS-4R (D), DT-4R/RS-5R (E) systems, and a circuit to determine the TRP length distribution (F). The light yellow domain in TRP indicates the toehold for the subsequent strand displacement. 83x153mm (300 x 300 DPI)


Page 16 of 18

Page 17 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. (A) Values of FA/FD for TRPs-closed DT-nR tweezer upon the introduction of RS-(n+1)R, and (B) amount proportion of TRPs with different numbers of repeats. 83x33mm (300 x 300 DPI)



Table of Contents artwork 84x47mm (300 x 300 DPI)


Page 18 of 18

A Smart DNA Tweezer for Detection of Human ... - ACS Publications

Recommend Documents