Probing Cellular Molecules with PolyA-Based Engineered Aptamer

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Probing Cellular Molecules with PolyAbased Engineered Aptamer Nanobeacon Lizhen Chen, Jie Chao, Xiangmeng Qu, Hongbo Zhang, Dan Zhu, Shao Su, Ali Aldalbahi, Lianhui Wang, and Hao Pei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16764 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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ACS Applied Materials & Interfaces

Probing Cellular Molecules with PolyA-based Engineered Aptamer Nanobeacon Lizhen Chen , Jie Chao , Xiangmeng Qu *, Hongbo Zhang#, Dan Zhu , Shao Su , Ali Aldalbahi †

‖

†

＆

†

‖

‖

, Lianhui Wang‖*, Hao Pei†*

Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry

and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, P. R. China ‖

Institute of Advanced Materials, Nanjing University of Posts and Telecommunications, Nanjing 210023, P. R. China #Division

of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland

＆

Chemistry Department, King Saud University, Riyadh 11451, Saudi Arabia

KEYWORDS: Nanobeacon, Aptamer, Spherical Nucleic Acid, Cellular analysis, Surface Engineering, Self-assembly

ACS Paragon Plus Environment

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ABSTRACT: Adenosine triphosphate (ATP) is a central metabolite that is of critical importance in many cellular processes. The development of sensitive and selective methods for the detection of ATP level in vivo is crucial in diagnostic and theranostic applications. In this work, we have developed a polyA-based aptamer nanobeacon (PAaptNB) with improved efficiency and speed of ATP analysis. We found that the dissociation constants and competitive binding kinetics of the PAaptNB could be programmably regulated by adjusting the polyA length. When the polyA length reached to 30 bases, a 10 µM detection limit for ATP assay with PAaptNB can be achieved (~10-fold improvement compared with the conventional thiol-based aptamer nanobeacon). The feasibility of the PAaptNB using for in vivo assay was further demonstrated by imaging intracellular ATP molecules. This study provides a new strategy to construct highefficiency and high-speed biosensors for cellular molecules analysis, which holds great potential in bioanalysis and theranostic applications.

INTRODUCTION Adenosine triphosphate (ATP) works as cellular energy currency in living cells, which is involved in many biological processes, such as cellular respiration and ion transportation.1 The concentration change of ATP in cells is closely related to many diseases, for instance, malignant tumors2,

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and Parkinson's diseases.4 Therefore, the ATP determination in cells is of great

importance in diagnosis and biochemical research. To this end, many approaches based on aptamers have been developed, including fluorescence spectroscopy,5-11 electrochemistry,12-17 chemiluminescence spectroscopy,18-20 and colorimetry.21-25 Among them, there is now


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considerable interest in applying fluorescent aptasenor26 for the ATP detection due to its low cost, high sensitivity, and good selectivity.27 Spherical nucleic acid (SNA) is one type of synthetic DNA nanostructures typically consisting of gold nanoparticle (AuNP) cores densely functionalized with DNA shells. Since the initial seminal work,28 SNAs have attracted worldwide interest as efficient agents in biosensor (in vitro/vivo), therapeutic, gene transfection, and regulation.29-34 DNA hybridization between DNA strands anchored on the surface of AuNPs and complementary oligonucleotides are crucial steps for SNA-based sensing systems. For instance, a recent work by Mirkin et al.35 reported a systematic study on the DNA hybridization of SNAs through thermodynamic quantification of binding events between free oligonucleotides and partially pre-hybridized SNAs. Their study reveals that DNA hybridization is affected by the collective contributions of surface density, charge repulsion, and strand conformation. On the basis of this mechanistic understanding, a more effective SNA with nearly 100% hybridization efficiency has been developed.35 As a result of SNA’s important role in many biological applications, it is therefore of great scientific and technological interest to programmably engineer the nanointerface with the aim to regulate the SNA-based sensor activity and construct high-efficiency and high-speed sensing systems. Herein, we report the use of polyA-based SNA aptamer nanobeacon (PAaptNB) that are spatially functionalized with rationally designed poly adenine (polyA) diblock oligonucleotides, through which we achieved programmable engineering of PAaptNB for ATP assay. We have previously demonstrated that polyA can serve as an anchoring block for preferential binding with the AuNP surface, which provides effective means to systematically modulate the lateral spacing and surface density of DNA on AuNPs by simply adjusting the length of the polyA block.36-38 Here our designed polyA diblock oligonucleotides contained a polyA block for anchoring onto


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the AuNP surface and a functional block for the ATP recognition, resulting in a “turn-on”39, 40 fluorescence detection of ATP (Figure 1). Compared to the conventional thiolated SNA based aptamer nanobeacon, its detection limit was significantly reduced (~10-fold improvement of sensitivity), which is sensitive enough to detect the ATP levels in different cellular compartments and monitor the dynamics of ATP in real-time at single cell level.41, 42 In addition, the PAaptNB performance can be programmably regulated by tuning the length of the polyA block, thus significantly extend the detection scale. EXPERIMENTAL SECTION Chemicals and Materials. All DNA oligonucleotides were synthesized and HPLC purified by Sangon Biotechnology Inc. (Shanghai, China). The sequences of the involved oligonucleotides are listed in Table 1. ATP, CTP, GTP, and UTP were all obtained from Sangon Biotechnology Inc. (Shanghai, China). Hydrogen tetrachoroaurate (III) (HAuCl4ˑ4H2O, 99.99%) was purchased from China National Pharmaceutical Group Corporation. Ultrapure water was obtained through a Millipore filtration system. Table 1 Sequences of the oligonucleotides Names

Sequences (5’-3’)

reporter

ACC TTC CTC CGC AAT ACT-Cy3

Thiol-P

SH-T10 ACC TGG GGG AGT ATT GCG GAG GAA GGT

PolyA10-P

A10 ACC TGG GGG AGT ATT GCG GAG GAA GGT

PolyA20-P


PolyA30-P


PolyA40-P


PolyA50-P



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Preparation of PAaptNB. The AuNPs of 13 nm were synthesized by citrate reduction of HAuCl4 as reported elsewhere43. In brief, 1 % trisodium citrate solution was added into a boiling, rapidly stirred solution of 1% HAuCl4. The solution was kept boiling and stirred for 20 min, and then cooled to room temperature. The prepared AuNPs were stored at 4 °C. The AuNPs were then functionalized with thiol-DNA or polyA-DNA following the previously reported protocols38,

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, respectively. In brief, 10 µL thiol-DNA (Thiol-P) or polyA DNA

(polyA10-P, polyA20-P, polyA30-P, polyA40-P, and polyA50-P) were added to 100 µL AuNPs solution (10 nM) to reach a final concentration of 2 µM DNA. Then a 2 µL of citrate-HCl buffer (500 mM, pH 3.0) was added into the mixture, and incubated at room temperature for 10 minutes. After that, the unbounded DNA was removed by three rounds of centrifugation (12000 rpm, 20 minutes). The supernatant was decanted and the DNA modified AuNPs were rinsed with equal volume of 100 mM of sodium phosphate buffer (pH 7.4) with 0.3 M of NaCl. Finally, the thiol-DNA (or polyA-DNA) modified AuNP conjugates were resuspended in an equal volume of stock solution (25 mM Tris-HCl buffer, pH 8.2, 300 mM NaCl) and stored at 4 °C. Fluorescent ATP assays in buffer. The Cy3-labeled ssDNA reporter solution was prepared in a stock solution (25 mM Tris-HCl buffer, pH 8.2, 300 mM NaCl, final concentration was 1 µM) and added into thiolated-DNA (or polyA-DNA) modified AuNPs solution (final concentration was 5 nM). The mixture was then incubated overnight at 37 °C in dark. The precipitate was then cleaned three times to remove the free ssDNA reporter via centrifugation at 12000 rpm for 20 min, and was finally resuspended in an equal volume of stock solution. For the ATP detection, solutions of varying concentrations of ATP were prepared in a stock solution (25 mM Tris-HCl buffer, pH 8.2, 300 mM NaCl) and then added into solution containing hybridized DNA modified AuNPs and Cy3-labeled ssDNA reporter, the final


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concentration ranging of ATP from 0 to 100 µM. The mixture was incubated at 37 °C for 30 min in dark, the fluorescence of which was measured afterwards using a fluorescence spectrometer (F-7000, Hitachi, Tokyo, Japan) at 515 nm excitation. Live-Cell Imaging of ATP with PAaptNB. Mouse L929 fibroblastic cells were cultured in DMEM medium, supplied with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. After 24 h attachment, the cells were washed with 1× PBS and replaced with fresh DMEM medium. 5 nM of aptamer nanobeacon was added and incubated with the cells for 4 h, subsequently the cells were washed three times with 1× PBS. 1 µM Cy3-labeled reporter was chosen as control. The intracellular fluorescence signals of the cells were observed with an inverted fluorescence microscope (BDS200-FL, Chongqing Optec Instrument Co., Ltd). In vitro cytotoxicity assay. Mouse L929 fibroblastic cells were cleaved by trypsin (HyClone) and seeded into 96-well (104 cells per well) tissue culture plates. After 24 h incubation, the cells were washed with 1× PBS and replaced with fresh DMEM medium containing PAaptNB at various concentrations of 0.1 nM, 1 nM, 5 nM and 10 nM, respectively. Untreated cells were set as a control. After 4 h incubation, the PAaptNB containing solution was removed and incubated in fresh medium containing 2% fetal bovine serum for another 24 h. For the cytotoxicity assay, the cells were washed and then incubated with 100 µL medium containing 10% of CCK-8 for 2 h. The optical densities (OD) of the mixtures were measured with a Microplate Reader at 450 nm. RESULTS AND DISCUSSION Figure 1 illustrates the working principle of programmable engineering of PAaptNB activity by regulating the length of polyA block. We first constructed a series of PAaptNB by employing polyA diblock oligonucleotides, in which polyA with programmable length ranging from 10, 20,


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30, 40 to 50 bases was used as the anchoring block on the AuNP surface and the ATP aptamer was designed as the appended recognition block that was complementary with the Cy3-labeled single strand DNA (ssDNA) reporter, ensuring the closed proximity of fluorophores to the AuNP surface upon hybridization. Here AuNPs with 13 nm diameter were selected due to the ease of synthesis and sufficient size for the effective fluorescent quenching. Figure S1 displays the particle size distribution of polyA30 tailed diblock DNA strand functionalized AuNPs (polyA30AuNPs) measured from TEM images, and the average diameter was determined as ~13 nm. In the presence of ATP molecules, the ATP aptamer formed a more stable hairpin structure on the AuNP surface and the ssDNA reporter was released to restore the fluorescence of Cy3, forming the basis for a sensitive and selective fluorescence “turn-on” assay of ATP. To verify the tuning effect, we then quantified the surface density of polyA or thiol tailed ATP aptamer on AuNPs by using a fluorescence based method.45 Indeed, as shown in Figure S2, we observed that the surface density of the ATP aptamer was programmably engineered, which decreased along with the increase of the polyA block length. In contrast, the surface density of thiolated ATP aptamer was determined to be 56 per AuNP, which was ~3.2-folds higher than that of polyA50 tailed ATP aptamer. We also carried out gel electrophoresis to further verify the tuning of surface density. Figure 2 shows that the migration rate of polyA-AuNPs gradually decreased as the polyA length was increased from polyA10 to polyA50, and was overall faster than thiolatedDNA-AuNPs. Previous studies have pointed out that the migration rate in gel electrophoresis is generally inversely related to the number of DNA strands on AuNPs.36 We thus concluded that the number of DNA strands per AuNPs, hence the lateral spacing and surface density of DNA strands were decreased with the increase of polyA block length. (Schematic illustration in Figure S2)


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Figure 1. Schematic illustrating that the activity of PAaptNB can be programmably engineered by tuning the length of polyA anchoring block.

Figure 2. Agarose gel electrophoresis images showing the increased migration rate with the increase of polyA length. L1: Citrate modified AuNPs, L2: Thiolated-DNA-AuNPs conjugates. We then test the feasibility of our PAaptNB in the ATP detection by using a displacementbased fluorescence method. The curve in Figure 3a shows the fluorescence spectrum of the free Cy3-labeled ssDNA reporter solution upon the excitation at 515 nm. After the addition of polyA30-AuNPs, the fluorescence decreased significantly, consistent with the fact that AuNPs are efficient quenchers (curve b in Figure 3a). We estimated that approximately 85% of the fluorescence of the Cy3-labeled ssDNA reporter solution was quenched by AuNPs, indicating an


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efficient hybridization between the Cy3-labeled ssDNA reporter and the recognition block (ATP aptamer) on the surface of AuNPs. After the addition of ATP, the ATP aptamer formed a more stable hairpin structure and the ssDNA reporter was released to restore the fluorescence of Cy3 (curve c in Figure 3a). Based on the fluorescence intensities of curves b and c, we estimated that approximately 80% of the Cy3-labeled ssDNA reporter was released from the AuNP surfaces in the presence of ATP, indicating an effective fluorescence “turn-on” phenomena. We further evaluated the fluorescence intensity of the released reporter from SNA with different length of polyA. As shown in Figure 3b, polyA30-AuNPs conjugates showed exceptional release ability of reporter ssDNA strand.

Figure 3. (a) The fluorescence signal of free reporter (1 µM Cy3-labled ssDNA, curve a), hybridization with polyA30-AuNPs conjugates (5 nM AuNPs, curve b) or in the presence of 100 µM ATP (curve c). (b) The fluorescence signal gain produced by different polyA length-AuNPs conjugates with 1 µM Cy3-labled ssDNA reporter in the presence of 100 µM ATP.

With the ability to quantitatively analyze competitive binding of ssDNA reporter and ATP to the ATP aptamer on the surface of SNA based on different length of polyA tailed ATP aptamer in the presence of ATP (100 µM). We then investigated the apparent dissociation constants (Kd, taken as the ATP concentration that induces half maximal fluorescence intensity change)

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and


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the kinetics of competitive binding (turnover number Kcat, the number of substrate molecules turned over per polyA-based nanobeacon per second). As shown in Figure 4a, PAaptNB demonstrated a higher apparent dissociation constants than that of thiolated ATP aptamer. More importantly, with increasing polyA length, the apparent dissociation constants was programmably enhanced (for instance, ~546 µM for polyA10 vs. ~421 µM for polyA50, corresponding to ~1.3-fold improvement), indicating that the binding affinity was heavily dependent on the surface density of ATP aptamer probes. In contrast, the Kd of the polyA50 tailed ATP aptamer on AuNP surface to ATP was improved ~1.42-fold compared to that of individual ATP aptamer in a solution system (421 µM vs. ~600 µM46), this result was consistent with previous reports47, and the Kd of the polyA50 tailed ATP aptamer on AuNP surface to ATP was 42-fold larger than the Kd deviation from the original ATP-binding aptamer measured in vitro selection (~10 µM).48 We then investigated the release kinetics of Cy3-labeled ssDNA reporter by monitoring the fluorescence enhancement (F/F0) with time in the presence of 100 µM ATP (Figure 4b). We note that the release efficiency of polyA-AuNPs was greatly improved compared to that of thiolated-DNA-AuNPs. In parallel, we investigated the turnover number (Kcat, kinetic parameters) of Cy3-labeled ssDNA reporter on AuNPs in the presence of 100 µM ATP. The turnover number for duplex dissociation gradually increased with the increase of polyA length (Figure 4c). The kinetics result also revealed that the release of Cy3-labeled ssDNA reporter became more favorable along with the increase of polyA length, thus resulting in an improved net signal gain for the ATP detection. Our previous study has shown that the full coverage of polyA blocks the nonspecific adsorption of DNA strands on AuNP surface and facilitates the appended recognition block a better upright conformation for hybridization.38 Thus the improved hybridization capability with the increase of polyA length may be attributed to the


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elimination of steric hindrance among the recognition block (ATP aptamer), Cy3-labeled ssDNA reporter, and ATP molecules. This is also in consistent with the enhanced enzyme activity observed on polyA tailed DNAzyme based SNAzyme.49 The improved hybridization capability suggests that a more sensitive sensing platform for the ATP detection can be constructed using polyA-AuNPs.

Figure 4. (a) The constants of dissociation constants (Kd) varies with the length of polyA block. (b) Fluorescence enhancement (F/F0) with time, where F is the fluorescence intensity of each sample at time t in the presence of 100 µM ATP and F0 is the initial fluorescence intensity in the absence of ATP. (c) The turnover number (Kcat, kinetic parameters) varies with the length of polyA block. The concentration of Cy3-labeled ssDNA reporter was 1 µM. After understanding this surface density dependent hybridization process, we further evaluated the PAaptNB performance with the polyA-length regulation. The assay activity can be programmed by simply regulating the length of polyA (Figure 5b-f). The green bars indicate the region of detection, in which the signals can be detectable but not quantifiable.50 Specifically, with the increase in the polyA length, the detection limits decreased from 100 µM (polyA10) to 10 µM (polyA30) (Figure 5g). Thus with this precise surface density regulation, the detection sensitivity was improved 10-fold. The thiolated DNA based ATP aptamer nanobeacon was also


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tested as a comparison, and showed same detection sensitivity compared to polyA10-AuNPs (100 µM vs. 100 µM) (Figures 5b and a). It is worth noting that the polyA30 based probe showed a high sensitivity with a detention limit of 10 µM, which was the same magnitude compared to previously reported multicolor nanobeacons (2 µM).51 We then investigated the selectivity of the polyA30-AuNPs based aptamer nanobeacon towards different ATP analogues. As shown in Figure 5h, the assay could effectively distinguish ATP from its three analogues, CTP, GTP, and UTP. The above results demonstrated that the newly designed polyA-AuNPs based aptamer nanobeacon possessed high sensitivity and selectivity towards ATP targets, which has great potential in clinical diagnosis.

Figure 5. The fluorescence measurements in the presence of ATP at various concentrations (10 µM to 3 mΜ) employing (a) thiolated-DNA-AuNPs and (b-f) polyA10 to polyA50, respectively. The green bars indicate the region of detection. (g) Detection limits can be programmed by tuning the polyA length. (h) Selectivity of the polyA30-AuNPs based ATP assay over UTP, GTP, and CTP (all at 100 µM).


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Figure 6. (a) Images of mouse L929 fibroblastic cell after incubation with 5 nM Cy3-labeled reporter/ATP aptamer nanobeacon (left) and 1 µM Cy3-labeled reporter (right) for 4 h at 37 °C. Scale bar: 20 µm. (b) Viability (measured by CCK-8 assay) of mouse L929 fibroblastic cells incubated for 4 h with different concentrations of polyA30-SNA based aptamer nanobeacon. ATP aptamer immobilized AuNPs had been demonstrated to specifically and sensitively detect extracellular ATP. To evaluate the ability of PAaptNB on intracellular ATP detection, the ATP aptamer nanobeacon was incubated with mouse L929 fibroblastic cells for 4 h, and monitored with inverted fluorescent microscope. Free Cy3-labeled reporter without SNAs were selected as control. As shown in Figure 6a, the cells incubated with ATP aptamer showed clear intracellular Cy3 signal and in contrast no signal was observed in the cells incubated with Cy3-reporter. Consequently, we believe that the intracellular signal in Figure 6a was due to SNAs’ ATP sensing. At last, the biocompatibility of PAaptNB was accessed by using the Cell Counting Kit-8 (CCK-8). As shown in Figure 6b, no significant variability of mouse L929 fibroblastic cells were observed in the presence of as high as 10 nM polyA30 based SNA, which indicated that the ATP aptamer nanobeacon was reliable for intracellular measurement of ATP in mouse L929 fibroblastic cells without causing adverse toxic effect. CONCLUSION


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In this work, we presented an aptamer nanobeacon for cellular molecules with programmably engineered assay activity. Our nanofabrication strategy employed the rational design of the polyA tailed ATP recognition diblock oligonucleotides, which eliminated the steric hindrance effect that suppressed the surface hybridization on AuNPs. This diblock oligonucleotides strategy results in an ATP aptamer nanobeacon with high and tunable sensing performance by adjusting the length of polyA block. In addition, we envisage that this design will open new horizons and bring insights for developing other SNA based sensors for detection of other molecules with varies molecular weights. We thus believe that this study will not only aid understanding of the behaviors of biomolecules at the nanointerface of SNA but also provide new insight into the design of SNA for applications in bioanalysis, molecular diagnostics, and therapeutics.

ASSOCIATED CONTENT Supporting Information. Surface densities of PAaptNB with different polyA length; Selectivity of PAaptNB towards ADP and AMP. AUTHOR INFORMATION Corresponding Author * E-mails: [email protected]; * E-mails: [email protected] ; * E-mails: [email protected] ; Author Contributions L.C. and J.C. contributed equally to the work.


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ACKNOWLEDGMENT This work was supported by Shanghai Pujiang Program (16PJ1402700), the National Basic Research Program (973 Program 2012CB932600, 2013CB933802), National Natural Science Foundation of China (grant numbers 21305151, 21422508, 31470960, 21373260, 2160508, 791123037), China Postdoctoral Science Foundation (2015M581565). Ali Aldalbahi would like to extend his sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this work (RG-1436-005). The authors gratefully acknowledges the start-up funding from East China Normal University.

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Probing Cellular Molecules with PolyA-Based Engineered Aptamer

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