Research Article www.acsami.org
DNA Polymerase-Directed Hairpin Assembly for Targeted Drug Delivery and Amplified Biosensing Yingying Wang,†,§ Li-Ping Jiang,†,§ Shiwei Zhou,† Sai Bi,*,†,‡ and Jun-Jie Zhu*,† †
State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China ‡ Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Laboratory of Fiber Materials and Modern Textiles, the Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071, China S Supporting Information *
ABSTRACT: Due to the predictable conformation and programmable Watson−Crick base-pairing interactions, DNA has proven to be an attractive material to construct various nanostructures. Herein, we demonstrate a simple model of DNA polymerase-directed hairpin assembly (PDHA) to construct DNA nanoassemblies for versatile applications in biomedicine and biosensing. The system consists of only two hairpins, an initiator and a DNA polymerase. Upon addition of aptamer-linked initiator, the inert stems of the two hairpins are activated alternately under the direction of DNA polymerase, which thus grows into aptamer-tethered DNA nanoassemblies (AptNAs). Moreover, through incorporating fluorophores and drug-loading sites into the AptNAs, we have constructed multifunctional DNA nanoassemblies for targeted cancer therapy with high drug payloads and good biocompatibility. Interestingly, using the as-prepared AptNAs as building blocks, DNA nanohydrogels are selfassembled after centrifugation driven by liquid crystallization and dense packaging of DNA duplexes. Taking advantage of easy preparation and high loading capacity, the PDHAs are readily extended to the fabrication of a label-free biosensing platform, achieving amplified electrochemical detection of microRNA-21 (miR-21) with a detection limit as low as 0.75 fM and a dynamic range of 8 orders of magnitude. This biosensor also demonstrates excellent specificity to discriminate the target miR-21 from the control microRNAs and even the one-base mismatched one and further performs well in analyzing miR-21 in MCF-7 tumor cells. KEYWORDS: DNA nanoassembly, drug delivery, cancer cell, signal amplification, microRNA
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INTRODUCTION
tides. By the introduction of an initiator, the DNA oligomers are triggered and autonomously self-assembled into nanostructures, which can be further used as building blocks to construct micron- or even milli-scale structures.17,18 In recent advances in triggered assembly systems, DNA toehold-mediated strand-displacement reactions have been used to design complex autonomous behaviors and achieve kinetically controlled self-assembly of various DNA nanostructures.19−24 For example, hybridization chain reaction (HCR) is one of the most well-known strategies.25,26 In a HCR, two
DNA nanotechnology has emerged as a powerful tool to construct nanoscale structures and devices through the specificity of Watson−Crick base pairing, which has been widely explored for versatile applications, including biotechnology, biomedicine, and nanoelectronics.1−7 Owing to the advantages of sequence programmability, predictable local geometry, intrinsic functionalities, and good biocompatibility, DNA nanostructures exhibit superior performances to other nanoparticles and polymeric nanomaterials.8−15 As an important approach of construction of DNA nanostructures, initiator-triggered self-assembly systems have been extensively researched.16 Typically, a triggered assembly system is composed of a small number of short synthesized oligonucleo© XXXX American Chemical Society
Received: July 13, 2016 Accepted: September 21, 2016
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DOI: 10.1021/acsami.6b08597 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
double-distilled water. Finally, the solution was precipitated by centrifugation for the third time and dispersed in 10 μL of doubledistilled water. The resulting solution containing DNA nanohydrogels was stored at 4 °C for further use. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were carried out on a JEOL-2010 transmission electron microscope (JEOL, Japan) and an S-4800 scanning electron microscope (Hitachi Co., Japan), respectively. Drug Loading into AptNAs. Different volumes (0, 0.1, 0.5,1, 4, 10, and 20 μL) of AptNAs (0.5 μM) were added into 50 μL of doxorubicin (Dox) (10 μM). The final volume was supplemented to 120 μL with double-distilled water. The mixture was incubated at room temperature for 24 h. The fluorescence spectra of Dox after incubation with AptNAs were measured on a RF-5301PC fluorescence spectrometer (Shimadzu, Japan) by setting the excitation wavelength at 490 nm. Confocal Fluorescence Microcopy Imaging. Cancer cells were plated in a 35 mm confocal dish and grown to ∼80% confluency for 24 h before the experiments. Cells (106 cells/mL) were incubated with Dox or AptNA-Dox complexes for 2 h at 37 °C, followed by washing with 1 mL of phosphate buffer (pH 7.4) twice and suspension in 200 μL of RPMI 1640 medium before imaging. All cellular fluorescent images were collected on a TCS SP5 confocal laser microscope (Leica, Germany), with the following excitation wavelengths and emission filters: Dox, 488 nm laser line excitation, emission BP (580 ± 20) nm filter; FITC, 488 nm laser line excitation, emission BP (520 ± 20) nm filter. All images were digitized and analyzed by Leica Application Suite Advanced Fluorescence (LAS-AF) software. Cytotoxicity Assay. The cytotoxicities of free Dox, AptNAs, or AptNA−Dox complexes for each individual type of cell were determined by the Cell Counting Kit-8 (CCK-8) assay (Sangon Biotech Co., Ltd., Shanghai, China). Briefly, cells (5 × 104 cells per well) were treated with free Dox, AptNAs, or AptNA−Dox complexes in RPMI 1640 medium for 2 h, followed by precipitation through centrifugation. After removing 80% supernatant medium, fresh medium was added for further cell growth and incubated for 48 h. Then, 10 μL of CCK-8 solution was added to each well and incubated for 3 h. The absorbance was measured at 450 nm with a 680 microplate reader (Bio-Rad, U.S.A.). Cell viability was calculated as described by the manufacturer. MiR-21-Triggered Assembly of DNA Nanoassemblies. The DNA species dumbbell probe (DP), HP-A′, and HP-B′ were treated by a quick annealing process before use (heated to 90 °C for 5 min and then allowed to cool to room temperature for 2 h). DP (10 nM, 10 μL) and 10 μL of miR-21 with different concentrations (or other miRNAs, that is, miR-141, miR-let-7d, and one-base mismatched miR21) were incubated for 1 h to obtain the DP-miRNA hybrid. Then, 8 μL of DP-miRNA hybrid, 8 μL of HP-A′ (25 nM), 8 μL of HP-B′ (25 nM), 1 μL of Klenow exo-DNA polymerase (5 U), 4 μL of 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM DDT, pH 7.9), 8 μL of dNTPs (0.2 mM), 1 μL of RNase inhibitor (40 U), and 2 μL of double-distilled water were mixed to a final volume of 40 μL and incubated for 1 h at 37 °C. Fabrication of Electrochemical Biosensor. First, 200 μL of thiol-modified capture probe (CP) (0.5 μM) was treated with 1 μL of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, pH 5.2) for 1 h at room temperature to reduce residual disulfide bonds. Then, 5 μL of CP (0.5 μM) was added to the surface of a pretreated gold working electrode and incubated for 16 h at 4 °C. The resulting electrode was washed with water twice and subsequently immersed into 6-mercapto1-hexanol (MCH) solution (1 mM) for 1 h. After washing with water twice, 10 μL of the products that were prepared via the process detailed in the Experimental Section were dropped on the electrode surface and incubated for 3 h at 37 °C, followed by washing with 0.1 M phosphate-buffered saline (PBS) buffer containing 0.5 M NaCl (pH 7.4). Finally, the electrochemical measurement was performed on a CHI 660D workstation (CH Instruments Inc., Shanghai, China) using a conventional three-electrode system: an Ag/AgCl (3 M KCl) as reference electrode, a Pt wire as a counter electrode, and the fabricated gold electrode as the working electrode. The electrochemical signal
DNA hairpins with sticky ends can be alternately opened upon being triggered by a primary recognition event, which results in autonomous self-assembly of polymeric nanowires and is applied to signal amplification.27−29 In addition to linear polymerization, a variety of nonlinear versions have also been proposed based on toehold-mediated strand-displacement reactions, achieving programmable self-assembly of higher dimensional nanostructures, such as multibranched DNA junctions,30 dendritic nanostructures,31−34 or even polyhedrons.35 However, to achieve these complex structures, the components involved in the systems, either DNA hairpins or double-stranded DNA substrates, are relatively complicated, and rational design and optimization of the sequences become restrictive and difficult. Alternatively, through acting on the DNA strands, enzymes can also control the dynamic behavior of DNA nanostructures.36−40 Recently, Garg, Reif, and co-workers proposed a directed polymerase activation method to self-assemble onedimensional DNA tiles.41 In this polymerase-activated system, no overhang was needed for the initial reactants, which efficiently avoided complicated design in sequences. They have demonstrated two different assembly pathways, a simultaneous assembly and stepwise activated assembly, in which the onedimensional assemblies are formed from the protected tiles that are activated via a DNA polymerase to undergo a linear assembly simultaneously and are activated sequentially one after another, respectively. The development of a simple and robust platform to construct complex and functional nanostructures with higher dimensions is still in demand. Herein, we present a polymerase-directed hairpin assembly (PDHA) strategy to address this challenge. The system consists of only two DNA hairpins, an initiator and a DNA polymerase. Upon addition of aptamer-modified initiator, aptamer-tethered DNA nanoassemblies (AptNAs) are assembled in a cascaded manner. By rational design, the AptNAs can be readily incorporated with multifunctional moieties, such as bioimaging agents and drug-loading sites, achieving specific recognition of cancer cells, bioimaging, and targeted drug delivery. Moreover, DNA nanohydrogels are self-assembled using the AptNAs as building blocks that are driven by liquid crystallization and dense packaging of DNA duplexes. In addition, the PDHA strategies are readily extended to amplified electrochemical detection of microRNA (miRNA), demonstrating versatile applications of the proposed assembly system not only in biomedicine for targeted cancer theranostics but also in biosensing for sensitive amplification detection.
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EXPERIMENTAL SECTION
Synthesis of Aptamer-Tethered DNA Nanoassemblies. This system consists of three DNA species, sgc8-initiator, HP-A, and HP-B, which were treated by a “quick annealing” process before use (heated to 90 °C for 5 min and then allowed to cool to room temperature for 2 h). HP-A (25 μM, 8 μL), 8 μL of HP-B (25 μM), 8 μL of sgc8-initiator (5 μM), 2 μL of Klenow exo-DNA polymerase (10 U), 4 μL of 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol (DTT), pH 7.9), 8 μL of deoxynucleotide triphosphates (dNTPs) (20 mM), and 2 μL of double-distilled water were mixed to a final volume of 40 μL and incubated for 1 h at 37 °C. The resulting AptNAs (1 μM) were stored at 4 °C for further use. Preparation of DNA Nanohydrogels. AptNAs solution (80 μL) was precipitated by centrifugation (18 000 rpm for 30 min at 4 °C) and washed with 40 μL of double-distilled water. Then, the solution was precipitated by centrifugation again and washed with 20 μL of B
DOI: 10.1021/acsami.6b08597 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Scheme 1. (a) Secondary Structure Mechanism and (b) Reaction Graph of PDHA for the Construction of AptNAs
was recorded with differential pulse voltammetry (DPV) by scanning from +0.2 to −0.6 V (versus Ag/AgCl) in 5 mL of 10 mM Tris-HCl buffer (pH 7.4) containing 5 μM [Ru(NH3)6]3+, which was degassed with nitrogen for 30 min. The experiment parameters are listed as follows: initial potential, +0.2 V; final potential, −0.6 V; amplitude, 0.05 V; pulse width, 0.2 s; sample width, 0.01 s; pulse period, 0.5 s; quiet time, 2 s; sensitivity (C or A/V), 1e−5 A/V.
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open or a solid triangle/circle, respectively. The initial conditions of this program are described via the state of each port in the reaction graph. The execution of this reaction graph is depicted as Scheme S1 in Supporting Information. The reaction pathways are verified by native polyacrylamide gel electrophoresis (PAGE) (Figure S2). Although side-products can be observed in the PAGE results (Figure S2, lanes 4−7), they have no influence on the assembly process because the AptNAs with relatively high molecular weight can only be triggered and assembled by initiator (lane 7). Characterization of AptNAs. Although the DNA nanoassemblies can be scaled up through the PDHA in theory, the rate of assembly might decrease with assembly size due to the steric hindrances not only from the hybridization between primer and hairpin but also from the DNA polymerase subunit to attach to the 3′-end of the primer and extend another copy of hairpin.41 In this study, “spacer” domains with poly-T of 15 nt (denoted as domain s in Scheme 1) are introduced to reduce the steric hindrances. To confirm this hypothesis, the influence of the ratio of sgc8-initiator to hairpins on the size of AptNAs is investigated by transmission electron microscopy (TEM) (Figure 1). A certain concentration of sgc8-initiator is added to a series of increasing concentrations of HP-A and HP-B (1:1) to accomplish different molar ratios, that is, 1:5:5, 1:10:10, and 1:20:20. In contrast to unassembled hairpins in the absence of initiator and even in the presence of Klenow exo-polymerase (Figure 1a), nanoassemblies are easily observed upon the introduction of sgc8-initiator into the PDHA system (Figure 1b−d). In addition, owing to the steric hindrances, the sizes of the DNA nanoassemblies are almost unchanged by varying the ratio of sgc8-initiator to hairpins, which demonstrate the average diameter of 40 nm. Although different ratios of hairpins to sgc8-initiator lead to the formation of AptNAs having similar sizes, it is obvious that, when the ratio is at 5:5:1, all the hairpins are consumed for the assembly (Figure 1b). Thus, a hairpin-to-sgc8-initiator molar ratio of 5:5:1 is enough for the PDHA system to assemble AptNAs and is used
RESULTS AND DISCUSSION
Construction of Aptamer-Tethered DNA Nanoassembly. The AptNAs are constructed through polymerase-directed hairpin assembly (PDHA) as illustrated in Scheme 1a. Two hairpin monomers, HP-A and HP-B, are designed with a stem length of 21 nucleotide (nt) and a loop of 42 nt. Thus, in the absence of initiator, HP-A and HP-B metastably coexist with each other. To endow the DNA nanoassemblies with specific recognition ability for target cancer cells, aptamer is modified on the 5′-end of initiator. As a proof of concept, aptamer sgc8, which can bind to human protein tyrosine kinase 7 (PTK7) that are overexpressed on the CCRF-CEM cell membrane but not on Ramos cells,42,43 is chosen as a model. Upon addition of sgc8-initiator, it binds to the loop domain b of HP-A (Scheme 1a, step 1), and the DNA polymerase, Klenow exo-polymerase that retains 5′ to 3′ polymerase activity but has lost 5′ to 3′ and 3′ to 5′ exonuclease activities used in this study, extends the strand using single-stranded initiator b* as primer (Scheme 1a, step 2). As a result, the single-stranded stem domain a* is liberated, which can bind to the loop domain a of HP-B (Scheme 1a, step 3). Similarly, the stem domain b* of HP-B is unraveled by DNA polymerase (Scheme 1a, step 4), which in turn activates HP-A (Scheme 1a, step 5), finally resulting in the autonomous assembly of AptNAs through PDHA in a cascaded manner (Scheme 1a, steps 6−8). The reactions are clarified using a reaction graph (Scheme 1b),30 in which HP-A and HPB are abstracted as a node with two ports: one triangular input port and one circular output port. Depending on whether the stem of hairpin is exposed (accessible) or sequestered (inaccessible), the state of each port is represented by an C
DOI: 10.1021/acsami.6b08597 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
treatment cannot cause a denaturation and renaturation of the as-formed AptNAs. To load Dox into AptNAs, Dox and AptNAs are mixed and stayed at room temperature to allow saturation of drug loading. Because the molar ratio of sgc8-initiator to hairpins in the initial reaction mixture is 1:5:5, each AptNA is able to provide about 240 Dox loading sites theoretically. After intercalation into AptNAs, Dox fluorescence can be dramatically quenched.45,46 Thus, the amount of Dox loading into the AptNAs can be calculated by fluorescence-quenching experiments. Upon a certain concentration of Dox (∼4 μM) loading into AptNAs, the fluorescence is quenched with increasing the amount of AptNAs (Figure 2). When the molar ratio of Dox to AptNAs is
Figure 1. TEM images of AptNAs that are assembled with different molar ratios of sgc8-initiator to hairpins (HP-A and HP-B) in the initial reaction mixture: (a) 0, (b) 1:5:5, (c) 1:10:10, and (d) 1:20:20. The final concentration of sgc8-initiator is 1 μM. The concentrations of hairpins are changed with different ratios accordingly. Sample (a) is obtained by mixing HP-A and HP-B with Klenow exo-polymerase and dNTPs in the absence of sgc8-initiator, in which the concentrations of HP-A and HP-B are 5 μM. The amount of Klenow exo-polymerase and the concentration of dNTPs used in all samples are 10 U and 4 mM, respectively. The reactions are carried out at 37 °C for 60 min. Scale bar: 100 nm. Figure 2. Fluorescence spectra of Dox (final concentration of ∼4 μM) with increasing equivalents of AptNAs.
in the subsequent experiments. It should be noted that, given the resolution of the TEM imaging, the side-products observed in PAGE results with small molecular weight are difficult to be seen by TEM. In addition, the polymerization time and the amount of Klenow exo-polymerase are optimized (Figure S3), and the size distributions of the AptNAs that are obtained under the optimum conditions (37 °C for 30 min with a ratio of sgc8-initiator to hairpins of 1:5:5) are investigated by dynamic light scattering (DLS) (Figure S4). Moreover, the stability of the AptNAs is examined. As shown in Figure S5, there is little change in particle sizes after treatment with 2 U/ mL DNase I for different times, indicating the enzymatic resistance of the proposed AptNAs under physiological conditions (the concentration of DNase in human blood is
