Facile Probe Design: Fluorescent Amphiphilic Nucleic Acid Probes

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A Facile Probe Design: Fluorescent Amphiphilic Nucleic Acid Probes without Quencher Providing Telomerase Activity Imaging inside Living Cells Yongmei Jia, Pengcheng Gao, Yuan Zhuang, Mao Miao, Xiaoding Lou, and Fan Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01777 • Publication Date (Web): 25 May 2016 Downloaded from http://pubs.acs.org on May 30, 2016

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Analytical Chemistry

A Facile Probe Design: Fluorescent Amphiphilic Nucleic Acid Probes without Quencher Providing Telomerase Activity Imaging inside Living Cells Yongmei Jia,†,‡ Pengcheng Gao,†,‡ Yuan Zhuang,†,‡ Mao Miao,† Xiaoding Lou,*,† and Fan Xia† †

Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, P. R. China *E-mail: [email protected].

ABSTRACT: Nowadays, the probe with fluorophore but no quencher is promising for its simple preparation, environmental friendliness and wide application scope. This study designs a new amphiphilic nucleic acid probe (ANAP) based on aggregationcaused quenching (ACQ) effect without any quencher. Upon binding with targets, the dispersion of hydrophobic part (conjugated fluorene, CF) in ANAP is enhanced as a signal-on model for proteins, nucleic acids and small molecules detection, or the aggregation of CF is enhanced as a signal-off model for ion detection. Meanwhile, due to the high specificity of ANAP, a one-step method is developed powerfully for monitoring the telomerase activity not only from the cell extracts but also from 50 clinic urine samples (positive results from 45 patients with bladder cancer and negative results from 5 healthy people). ANAPs can also readily enter into cells and exhibit a good performance for distinguishing natural tumor cells from the tumor cells pretreated by telomeraserelated drugs or normal cells. In contrast to our previous results (Anal. Chem. 2015, 87, 3890−3894), the present CF is a monomer which is just the structure unit of the previous fluorescent polymer. Since the accurate molecular structure and high DNA/CF ratio of the present CF, these advanced experiments obtain an easier preparation of probes, an improved sensitivity and specificity, and broader detectable targets.

Biosensors play a very important role in early disease diagnostics,1 food safety,2 and environmental monitoring.3 Among them, nucleic acid probes with fluorescence signals is promising for high affinity and low interference of their target.4−6 In 1996, Sanjay Tyagi and Fred Russell Kramer designed the molecular beacons (MBs) as hairpin-shaped oligonucleotide probes, which reported the existence of the specific DNA in solution.7 Upon binding to target DNA, the probe underwent a conformational change which restored the fluorescence of fluorophore in the quenched probe. Since then, researchers specifically detected varieties of targets, including proteins, nucleic acid strands, small molecules, and ions by using the molecular beacons.8−14 The distance between the fluorophores and the corresponding quencher groups can be precisely tuned by the bindings between nucleic acid probes and their specific targets, which induce either on model or off model of the fluorescence signals. The detection by using nucleic acid probe is sensitive, specific, speedy, accuracy and even automatic processes.15−19 In the past two decades, the combination of fluorophore and quencher group (such as nanorod, nanosheet, and nanowire)20−26 is a commonly used strategy for the inorganic or biological detection. However, in nucleic acid probes, the coexistence of both fluorophore and the quencher group, not only bring the inconvenience of organic synthesis, but also induce the difficulties to precisely control the distance between fluorophore and quencher group. All above limit the applications of the nucleic acid probes with fluorescence signals in practical duties, for example, point-ofcare testing (POCT).27,28 In this contribution, we design a new amphiphilic nucleic acid probe (ANAP), which is with only fluorophore but no

quencher. The ANAP is comprised of a hydrophobic fluorophore unit (conjugated fluorene, CF), a linker and a hydrophilic DNA primer (Figure 1a). The synthetic CF is a fluorescent molecule whose exciting wavelength is at 380 nm and emission wavelength is at 410 nm (Figure S1). In optimized aqueous solutions, the probe can form specific micelles due to the amphipathy of the hydrophobic CF core and the hydrophilic DNA shell (Figure 1b). The aggregation of CFs causes the quench of fluorescence, namely aggregation-caused quenching (ACQ) effect.29 In practice duties, upon binding with the targets, the dispersion of CFs is enhanced as a signal-on model, or the aggregation of CFs is enhanced as a signal-off model. This work is the follow-up of our previous paper (Anal. Chem. 2015, 87, 3890−3894). The fluorescent unit (CF) in this work is a monomer that is the structure unit of the fluorescent polymer in the previous work. Compared with the fluorescent polymer, the fluorescent monomer is not a polymeric compounds with high degree of dispersion of and possesses higher purity which facile the preparation and broaden the detectable targets (signal-on model for proteins, nucleic acids, small molecules and signaloff model for ions). Meanwhile, the molecular weight of the synthetic CF monomer is relatively small (MW= 718.9) in comparison with the DNA primer (MW=5523.6) (Figure S2), which effectively reduce the attractive force between CFs and enhance fluorescence efficiency in the dispersed state. By virtue of relatively small mass proportion of CFs, the ANAP presents a high specificity for monitoring the telomerase activity of 50 clinic urine samples (positive results from 45 bladder cancer patients and negative results from 5 healthy persons), and distinguishing natural tumor cells from the

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tumor cells pretreated by telomerase-related drug or normal cells. EXPERIMENTAL SECTION The synthesis route of hydrophobic fluorophores are on the basis of previous works,30−32 as shown in Scheme S1. The synthesis is confirmed successfully by 1HNMR (results in supporting information). Synthesis of 2, 7-bis(4-phenylcarboxylic acid methyl ester)-9, 9-di-n-octylfluorene (CP1). 2,7-bis(4phenylcarboxylic acid methyl ester)-9,9-di-n-octyl fluorene (CP1) is syntheized on the basis of the pervious report.33 3.18 g 2,7-Dibromo-9,9-di-n-octyl fluorene, 2.73 g 4carboxymethylphenylboronic acid and 18 mL K2CO3 aqueous solution (2 M) are dissolved in 36 mL THF in a Schlenk flask. After the solution is aerated by argon for 15 min, 200 mg tetrakis-(triphenylphosphine) palladium(0) is added to the solution. Then this mixture is heated for 18 h at 80 oC. The product is extracted and dulited into dichloromethane, rinsed by brine, and dried by MgSO4. Then the crude product is recrystallized by ethanol. The final pure diester presents as yellow shiny crystals. Synthesis of 2,7-bis(4-phenylcarboxylic acid)-9,9-di-noctylfluorene (CP2). 1.40 g CP1, 1.60 g KOH, 20 mL THF and 10 mL H2O are mixed and refluxed for 16 h, then concentrated HCl is used to acidified the mixture. The white solid is precipitated, and respectively risined with water and ethanol for three times, and then vacuum-dried. Synthesis of hydrophobic fluorophore in amphiphilic nucleic acid probe (CF). 800 mg CP2, 760 mg Nhydroxysuccinimide (NHS) and 180 mg dicyclohexylcarbodiimide (DCC) are dissolved in N,N’dimethylformamide (DMF, 16 mL). The mixture are stirred for 48 h at room temperature. After solvent evapration, silicagel column chromatography is used to further purified the crude product. Synthesis of the amphiphilic nucleic acid probe (ANAP). CF (0.25mg, 300 nmol) is dissolved in dry DMF (250 µL), then added to the hydrophilic DNA primer (10 OD). By adding 0.1 M sodium tetraborate buffer (pH = 8.5), the total volume is controlled as 500 µL . The solution is stirred overnight at room temperature. A high performance liquid chromatography (HPLC) method is used for purification of the product (ANAP). RESULTS AND DISCUSSION CF is synthesized via a Suzuki coupling reaction as shown in Scheme S1. In mass spectra (Figure S3 and Table S2), the strong peak (at 6245.7) is attributed to the amphiphilic nucleic acid probe (ANAP), which coincides with the calculated molecular weight (5523.6 (nucleic acid) + 718.9(fluorophore) = 6242.5). The identity and purity of the products are also verified by 1HNMR (experimental details in supporting information). In optimized aqueous solutions, due to the hydrophobic CF core and the hydrophilic DNA shell, the amphiphilic ANAPs aggregate into micelles (Figure S4), leading to the quench of fluorescence, which means the fluorescence intensity increase with the attenuation of aggregation.27,28 In a THF/H2O mixture, the fluorescence intensity increases with the proportion of THF, which is a good solvent of CFs but poor solvent of DNA primers (Figure S4). Meanwhile, when the hydrophilic properties of the ANAP enhanced, part of micelles collapse and the fluorescence

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liberates simultaneously (from Figure 1b to Figure 1c). DNA is the hydrophilic part of ANAP. The hydrophilicity of ANAP can be enhanced by simply increasing the DNA content, which is achieved by DNA extension in the present work. The DNA unit in ANAP conjunct with the DNA ligase and further extend by grafting the linker DNA (LDNA) with incremental base number. The products of ANAP are verified by gel electrophoresis analysis (Figure S5). The fluorescence intensity of ANAP gradually enhance with the base number of LDNA from 6 bases in LDNA0 to 60 bases in LDNA4 (Figure 1d). On the other hand, when the hydrophilic properties of the ANAP weaken, the aggregation of the fluorophores enhance and suppress the fluorescent emission (from Figure 1b to Figure 1e). As a sequence-independent nuclease, exonuclease I (Exo I) can catalyze the hydrolyzation of mononucleotides individually from the 3’ terminal of single stranded DNA. Thus, it is selected to digest DNA and reduce the hydrophilicity in ANAP. As shown in Figure 1f, by adding Exo I, the fluorescence signal of ANAP almost reduce to background within 20 minutes. With the clip of the DNA chain, the hydrophilicity of ANAP weaken that leads to the enhancement of the CF aggregations and the fluorescence quenching. The presence of Exo I have no influence to fluorescence signal of CF. All data demonstrate that the hydrophilic/hydrophobic properties of ANAP can be conveniently controlled by simply changing the relative content of DNA. This character is unique to other probes

Figure 1. (a) ANAP contains a hydrophobic fluorophore, a linker and a hydrophilic DNA part but without quencher, which formed micelles with DNA shell and CF core in optimized aqueous solution. From (b) to (c), the collapse of the micelles induced the increase of fluorescence (signal-on model). On the contrary, from (b) to (e), the micelles continued aggregate, inducing the decrease of fluorescence (signal-off model). (d) The fluorescence of ANAP increased with the DNA length. DNA sequences were shown in Table S1. (f) The DNA part in ANAP was digested by Exo I, decreasing the hydrophilic property of ANAP, reducing the fluorescence finally. Concentration of ANAP was 87 nM. Excitation wavelength was 340 nm (Figure S1). Error bars were calculated from three independent experiments.


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(Figure S6). The results above demonstrated that the fluorescence emission or quenching of the ANAP with the CF of small molecular weight can be regulated by adjusting its hydrophilicity by the telomerase extension or the Exo I digest of the hydrophilic DNA unit in the ANAP. Since the fluorescence emission of ANAP can be regulated by hydrophilic adjustment, we are inspired to apply the ANAP for monitoring protein biomarkers. Here, telomerase is choosen as a model for protein biomarkers. Telomerase is a reverse transcriptase which can add repeated fragments (TTAGGG)n to the end of chromosomes.34-42 Different assays have been designed for the detection of telomerase since 1985 when the telomerase was discovered. Three categories of the detections include: 1. The classical PCR based telomeric repeating amplification protocols, known as TRAPs;43 2. The biosensors based on varieties of nanoscalematerials;44−46 3. Isothermal detection methods.47−50 The above strategies achieve good sensitivity and specificity. However, most of them detected the telomerase in cell lysates as samples, which are incompetent to detect telomerase in real environments, especially in living cells. Recently, Ju’s group developed the nanoparticles based probes to detect the telomerase in living cells.51,52 However, the probe designs are still complicated, involving a nicked molecular beacon, nanoparticles and so on. The development of facile and robust assays for tracking of intracellular telomerase activity in situ is still of great importance. In the present work, on purpose of detecting telomerase, ANAP is designed by conjugating 5’ end of telomerase primer to CF (experimental details in supporting information). The mass proportion of DNA in the ANAP before telomerase extraction, related to the hydrophilicity of ANAP, isn’t high enough to trigger the unrestricted dispersion of CFs. Thus, the aggregation of hydrophobic CF is still favored leading to a low fluorescence signal. When adding telomerase, the telomerase primer conjugates with the nucleic fragments of telomerase as (TTAGGG)n, leading to extension of the hydrophilic unit in the ANAP (verified by gel electrophoresis analysis in Figure S7). The improvement of hydrophilicity initiate the enhancement of the dispersion of CFs and corresponding fluorescent intensity (Figure 2a). EJ cells are typical bladder cancer cells and with high expression of telomerase. With the number of EJ cells increment, the fluorescent intensity of the ANAP solution increase progressively (Figure 2b). Based on F+3σb (F, average fluorescence intensity of the sample blank. σb, standard deviation of the sample blank), the limit of detection (LOD),53 is 28 EJ cells/µL. The LOD of our method is comparable with other sensing assays for telomerase activity detection, which mostly require the costly instruments operated only in an analytical laboratory, such as surface plasmon resonance spectrometer,54 electro-chemistry assay,55 and PCR based assay56 (Table S3). When the number of EJ cells increases from 28 to 28000 cells, the emission of ANAP (87 nM) displays a hypsochromic band from 412 to 392 nm (Figure 2b and Figure 2). The fluorescence intensity enhancement remains the same when concentration of ANAP is 250 nM (Figure S8). The extension between the conjugated emissive units during the intermolecular interactions leads to the hypsochromic of emission,57,58 which also demonstrates the disassembly of ANAP aggregation in the presence of telomerase. In addition, after adding EJ cells, the fluorescence signal gradually increases and finally reaches a platform at approximately 60 minutes (Figure 2d). For the specificity, in

the presence of thrombin, inactive telomerase or BSA, the fluorescence intensity shows no distinction with the control sample (without telomerase) (Figure 2e). In order to illustrate the generality and reliability of our detection platform, noncancer cells are also employed (experimental details in supporting information). As shown in Figure 2f, we observes significant enhancements of fluorescence intensity for cancer cell extracts from EJ (bladder cancer cell), HeLa (cervical cancer cell), MCF-7 (breast cancer cell), A375 (melanoma cancer cell), HepG2 (liver cancer cell), and T24 (bladder cancer cell), however, relatively low signal for non-cancer cell extracts from HLEC (human lens epithelial cell). The results demonstrated that our assay offers a simple, non-invasive, reliable, sensitive and specific assay to detect the proteins extracted from the cells. In addition to the detection of telomerase in living cells, we further employ our method to detect crude extracts of patients’ with bladder cancer and normal people’s urine specimens. As shown in Figure 2g, the

Figure 2. (a) Schematic represented the telomerase detection method by using the binding regulation of the hydrophilic/hydrophobic properties of ANAP. (b) Fluorescence emission spectra in the presence of different number of EJ cells. (c) Corresponding emission wavelength vs. the logarithm of the EJ cell concentrations. (d) Time dependent fluorescence responses of our assay. (e) Histogram of fluorescence intensities in the presence of telomerase or various interfering proteins to investigate the specificity of ANAP. (f) Fluorescence responses to telomerase from 2800 cells of different kinds of cancer and non-cancer cell lines. The horizontal dashed line represents the threshold level. (g) Fluorescence responses to telomerase from 45 bladder cancer patients and 5 normal people. Reaction temperature was 37 oC.



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orange part is the fluorescence intensity for the 45 patients with bladder cancer. The green part is the fluorescence intensity for the 5 normal persons. The classification for cancer and healthy persons is clear according to the result above, that achieve the specific and sensitive detection of telomerase. In view of the good detection efficiency of the ANAP in solution, we further test its cellular uptake and fluorescence changes in living cells by using HeLa cells (cervical cancer cells) as a model (Figure 3a). To verify the non-cytotoxicity of ANAP to living cells, the MTT (3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide) assay is carried out. The result shows that the cell viabilities are more than 84% when 0.1, 1.0 and 5.0 µM ANAP are added to the culture medium for 36 h, indicating the low cytotocixity of ANAP (Figure S9). Moreover, the cytotoxicity of lipofectamine in the presence and absence of 5.0 µM ANAP is tested (Figure S10). The cell viability of cells with lipofectamine (77.2%) show no obvious difference to cells with lipofectamine and ANAP (80.8%). The results above indicate that ANAP transfection do not hamper the cell viability. Then Transfection is carried out by using 250 µL of Opti-Mem (Invitrogen) containing 8.33 µL lipofectamine 2000. Upon transfection, ANAP can be extended by intracellular telomerase at its 3’ end, which leads to the enhancement of hydrophilicity in ANAP. Thus, the fluorescence turns on. Reportedly, although most telomerase are in the nucleus, some telomerase are still present in the cytoplasm of the perinuclear area.51,59 Here, The telomerase in cytoplasm are choosen to be detected. The fluorescence detected in cells’ images are indeed existing in cytoplasm around the nucleus, which conforms to the reports. The HeLa cell transfected with ANAP displays a strong blue fluorescence (Figure 3b). When transfected with AZT an inhibitor for telomerase, only negligible fluorescence can be observed in the HeLa cells containing the ANAP (Figure 3c). A 3D surface projection of Z-stack images for HeLa cells treated with ANAP clearly shows the dyeing of cells (Figure 3d), while AZT-treated HeLa cells shows negligible emission (Figure S11). As shown in Figure S12a and S12b, after treating by AZT, the fluorescence intensity of HeLa cell containg ANAP decrease 3.22 times. As a control experiment, HLF (human lung fibroblast) cells as normal cells are choose to be transfected by ANAP. Both the HLF cell containing ANAP and the cells further treated by AZT exhibit a negligible fluorescence (Figure S12c and S12d), that verify the specific recognition of telomerase in cancer cells by using ANAP as probe. Additionally, the fluorescence intensity of the ANAP in HLF is 1.37 times higher than that in the AZTtreated HLF, which means AZT also inhibits the telomerase activity in HLF (Figure S12e). These results verify that ANAP has great potential in noninvasive tracking of intracellular telomerase activities, offering a platform for the screening of drugs related to telomerase. In consideration of the good performance for the protein biomarker detection, we are further encouraged to employ our ANAP detection platform to detect broader targets, including nucleic acids, small molecules and ions. Methods for real time, high sensitive nucleic acid detection are of vast scientific and clinic importance.60−63 As shown in Figure 4a, when the ANAP hybrid with its complementary DNA targets, the fluorescent intensity dramatically increases as 6.12 times (from “ANAP” bar to “Matched” bar) (Figure 4g). The LOD of ANAP towards DNA is 3 nM as shown in the calibration

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curve of target with different concentrations (Figure 4d). In contrast, when treated with non-complementary target, the change of fluorescent signal is negligible (from “ANAP” bar to “Mismatched” bar) (Figure 4g). The result above demonstrates that the ANAP platform could be used for DNA detection. The ANAP detection platform is also performed to detect small molecule by using ATP. As a well-known cellular energy source, ATP can drive various of biological processes.64 We synthesize 14-base and 13-base fragments corresponding to the 27-base of the ATP binding aptamer.65,66 The ANAPs are synthesized by covalent binding CF with the 5’ end of the 13-base DNA. While non-specific interaction occurs between the 3’ fragments and the 5’ fragments even in the absence of ATP,67 ATP can stabilize the associated complexes as shown in Figure 4b. Upon binding with the ATP, the hydrophilic unit in the ANAP and the corrseponding dispersion of CFs is enhanced. When the solution was challenged with ATP, fluorescence intensity increases. The fluorescence intensity increases with the concentration of ATP

Figure 3. (a) Scheme of the ANAP for in situ analysis of intracellular telomerase. (b, c) Confocal microscopy images of HeLa cells treated with (b) ANAP and (c) ANAP+AZT (inhibitor for the telomerase). Each series can be classified as the CF luminescent images (left), merged images (middle), and bright field (right), respectively. Scale bar: 50 µm. (d) A 3D surface projection of Zstack images for HeLa cells treated with ANAP. Scale bar: 20 µm.


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(Figure 4e). The LOD of ATP by using the ANAP with the designed DNA sequence as probe is 2.5×10-8 M. Furthermore, ANAP with the designed DNA sequence are certified to specifically detect ATP rather than other nucleoside triphosphate UTP, GTP and CTP (Figure 4h). Moreover, the designed ANAP performs well even in the cellular lysate, and corresponding changes in the fluorescence signal are observed in the cellular lysate, which presents the good specificity of the designed ANAP (Figure S13). All the above ANAP sensors work depending on the enhancement of hydrophilicity, defined as signal-on models. While the ANAP can also detect ions based on its signal-off model, owing to ACQ effect for the improvement of the CF aggregation. Mercury ions are chose as a model for ion detection, which are well-known toxic and highly harmful to human.68,69 As shown in Figure 4c, Hg2+ ions enhance the aggregation of ANAP by formation of T-Hg2+-T pairs. The fluorescent intensity decreases by virtues of ACQ effect in the presence of Hg2+. The LOD reach as low as 2.1×10-11 M (Figure 4f). Simultaneously, the ANAP present a detection of Hg2+ ions with high specificity according to a number of metal ions (Cu2+, Co2+, Pb2+, Ca2+, Mg2+, Zn2+) under identical conditions (Figure 4i).

as DNA primer, which effectively reduce the attractive force between CF fluorescent molecules and enhance fluorescence efficiency in the dispersed state. Owing to efficient aggregation or dispersion, the designed ANAP present a high sensitive and specificity which is applied for monitoring the telomerase activity of 50 clinic urine samples, and distinguishing tumor cells from the tumor cells pretreated by telomerase-related drugs or normal cells. Simultaneously, the present fluorescent unti (CF) is a monomer as the structure unit of the fluorescent polymer disscussed in our previous work.37 The advance of the present work is the facile preparation and the universally detectable targets, including proteins (telomerase), nucleic acids (DNA), small molecules (ATP) and metal ion (Hg2+), brought by using the fluorescent monomer.

SUPPORTING INFORMATION Detailed description of the experiments, the sensor fabrication, the characterizations of targets (telomerase, DNA, ATP, Hg2+) and additional figures are listed. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

Author Contributions ‡

Y.J., P.G., and Y.Z. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by National Basic Research Program of China (973 program, 2015CB932600, 2013CB933000), the National Natural Science Foundation of China (21525523, 21375042, 21574048, 21405054) and 1000 Young Talent (to Fan Xia). This Project is also supported by China Postdoctoral Science Foundation (2014M562010, 2015T80787).

REFERENCES

Figure 4. (a-c) Schematic representation of nucleic acids (DNA), small molecules (ATP) and inorganic ions (Hg2+) detection strategy using ANAP. (d-f) and (g-i): Calibration and specificity curves for detection DNA, ATP and Hg2+, respectively. Excitation wavelength was 340 nm. Error bars were obtained by three parallel experiments. Concentration of ANAP is 87 nM.

CONCLUSIONS In brief, we design an ANAP based on aggregation-caused quenching (ACQ) effect but no quencher. In the synthetic ANAP, the hydrophobic unit as fluorescent CF is of small molecular proportion in comparsion with the hydrophilic unit

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For TOC only


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