A Programmable Target-Initiated DNAzyme Walker Walking along a

Subscriber access provided by TULANE UNIVERSITY

Functional Inorganic Materials and Devices

A Programmable Target-Initiated DNAzyme Walker Walking along a Spatially Isolated and Highly Hybridizable Substrate Track on Nanoparticle Surface Ke Yang, Huizhen Wang, Ning Ma, Ming Zeng, Huaiqing Luo, and Dinggeng He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16408 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on December 2, 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.

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 27 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

ACS Applied Materials & Interfaces

A Programmable Target-Initiated DNAzyme Walker Walking along a Spatially Isolated and Highly Hybridizable Substrate Track on Nanoparticle Surface Ke Yang,*,† Huizhen Wang,⁋ Ning Ma,† Ming Zeng,† Huaiqing Luo,† and Dinggeng He*,‡,⁋,‖ †

Hunan Key Laboratory Cultivation Base of the Research and Development of Novel

Pharmaceutical Preparations, Department of Human Anatomy, Histology and Embryology, Changsha Medical University, Changsha 410219, China ‡

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences,

Hunan Normal University, Changsha 410081, China. ⁋

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and

Chemical Engineering, Hunan University, Changsha 410082, China. ‖

Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.

KEYWORDS: autonomous movement, DNA nanotechnology, DNAzyme, gold nanoparticle, molecular machine

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 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

Page 2 of 27

ABSTRACT: Synthetic DNA machines that operate on the nanoscale three-dimensional (3D) track have attracted rapidly increasing interest because of their potential in biocomputing, drug delivery, and biosensing applications. Current nanoscale 3D DNA tracks are typically created by self-assembling thiolated oligonucleotides at gold nanoparticles (AuNPs) surface via the strong Au-S chemistry. However, it remains challenging to accurately control the conformation and orientation of 3D DNA track on AuNPs surface and finely adjust the hybridization ability of 3D track. Herein we describe for the first time a poly adenine (polyA)-based, spatially isolated 3D DNA track, on which a target-initiated DNAzyme walker moves by a burnt-bridge mechanism with improved efficiency and processivity. PolyA serves as an anchoring block for preferential binding with AuNPs surface, and the appended substrate block adopts an upright conformation that favors the hybridization and subsequent DNAzyme-mediated cleavage. The operation of this target-initiated DNAzyme walker was monitored in real time and at single-particle level. We tested the cleavage efficiency of 3D substrates with various polyA block lengths, which displayed that the DNAzyme activity was remarkably improved as compared to thiol-based 3D track. We also explored bioanalytical applications of this DNAzyme nanomachine by movementtriggered cascade signal amplification.

INTRODUCTION Living organisms take advantage of the complicated molecular machines to perform various types of physiological functions such as intracellular transport, mechanical actuation and signal transduction.1-3 Inspired by nature, a large number of biomimetic DNA machines have been typically created that are propelled by strand displacement, DNAzymes and protein enzymes.4-7


2

Page 3 of 27 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


These artificial DNA machines perform stepwise walking autonomously and progressively along a prescriptive one-dimensional DNA footpaths8-11 or two-dimensional DNA origami track.12-14 Instructional strands are included precisely at specific positions of DNA tracks, enabling control of the walking direction of the DNA walkers. The engineered DNA machines have shown wide applications in biosensing,15-17 cargo transport,18,19 material assembly and synthesis20,21 and biocomputing.22 Recently, Ellington and co-workers showed the first example of stochastic DNA walker that walks randomly along a three-dimensional (3D) track on microparticle surface.23 The motion of DNA walker is well-confined at micrometer space. Afterwards, nanoscale 3D track has been established by employing the strong Au-S chemistry to assemble the thiolated oligonucleotides on AuNPs surface,15,24-29 which successfully achieves the random and stepwise movement of DNA walker at nanoscale space. Such a synthetic 3D DNA nanomachine has attracted great interest because of its potential in drug delivery15 and biosensing applications,24-26 particularly in living cells.27-29 Nevertheless, the Au−S chemistry used for the cross-linking of DNA strands and AuNPs is not able to accurately control the conformation and orientation of surface-tethered DNA strands,30 thus limiting the hybridization ability of 3D DNA tracks and the processivity of DNA nanomachines. Although the DNA conformation on AuNPs surface can be modulated by mercaptohexanol (MCH) backfilling to some extent,26 the MCH treatment probably decreases the stability of 3D DNA track and results in contamination and uncertainty in displacement.31 To address these issues, it is highly desirable to establish a AuNPs-based 3D track with accurate spatial control and tunable hybridization ability. Previous studies have demonstrated that polyA preferentially bind to Au surface with highadsorption affinity, even comparable to Au-S chemistry.32-34 Fan et al. employed this strategy for


3


Page 4 of 27

the simple synthesis of spatially controlled and highly hybridizable functionalization of DNAAuNP nanoconjugates.35-37 PolyA-mediated DNA assembly on AuNP is a desired surface modification strategy for constructing 3D DNA track with accurate spatial control and tunable hybridization ability. Recently, DNAzymes have been found with growing interest as driving mechanisms of DNA machines.6,27 The flexibility in regulating DNAzyme structure by encoding specific functional information in its base sequence turns DNAzyme into an ideal candidate for the development of programmable target-initiated DNAzyme walkers. Inspired by these works, we demonstrate here a spatially isolated and highly hybridizable 3D DNA track, on which the stochastic DNAzyme-based walker autonomously moves through a burnt-bridge mechanism with improved efficiency and processivity. The driving force is derived from unidirectional cleavage of 3D substrate track with the rationally designed DNAzyme that is initiated by various target molecules.

MATERIALS AND METHODS Preparation of Gold Nanoparticles (AuNPs). Colloidal AuNPs with the diameter of 18±2 nm were prepared.38 In brief, trisodium citrate solution (1%) was added to a boiling, rapidly stirred solution of HAuCl4 (HAuCl4•4H2O, 99.99%, Sigma-Aldrich). The mixture solution was stirred for 20 min, and then cooled to room temperature. The obtained citrate-stabilized AuNPs were stored at 4 °C before use. Preparation of polyA-DNA-AuNPs. Oligonucleotides used in this work were purchased from TaKaRa Inc. (Dalian, China), and the detailed DNA sequences are shown in Table S1. AuNPs were coated with polyA-DNA (A5, A10, A15, A20, A25). In brief, the prepared AuNPs


4

Page 5 of 27 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


(5 nM) were first treated with polyA-DNA in a molar ratio (polyA-DNA / AuNPs) of 200 for 16 h. The resulting solution was then added to 10 mM sodium phosphate buffer (pH 7.4) and 0.1 M NaCl, allowed to rest for 40 h. Subsequently, the particles were collected and washed three times with 10 mM sodium phosphate buffer (pH 7.4) to remove excess polyA-DNA. The obtained nanoconjugates were dispersed in phosphate buffer (pH 7.4, 10 mM sodium phosphate, 0.3 M NaCl). Quantification of DNA Surface Density on AuNPs. Quantity of polyA-DNA on AuNPs surface was determined by a fluorescence method based on the strand displacement. First, polyA-DNA used in this study was labelled with carboxyfluorescein (FAM) at 3’ end (See Table S1). FAM-labelled polyA-DNA was then adsorbed on AuNPs surface following the protocol outline above. Mercaptoethanol (MCH) with final concentration of 20 mM was added to the solution of fluorescently labelled polyA-DNA-AuNPs and incubated overnight with gentle shaking.35,39 Released polyA-DNA molecules were collected and the molar concentrations were determined by the fluorescence spectrometer (F-4500, Hitachi, Tokyo, Japan). Concentrations of AuNPs were calculated by the UV-Vis absorption spectra. TIRF Imaging and Analysis. All coverslips were washed before use. 22-mm square glass coverslips were sequentially sonicated for 30 min in household detergent, 30 min in acetone, and 30 min in absolute ethanol. The slides were then successively soaked in Piranha solution (H2SO4/30%H2O2) (v/v 3:1) for 30 min and sonicated for another 30 min, sonicated in HCl/30%H2O2/H2O (v/v/v 1:1:1) solution for 30 min, and sonicated in distilled water for 15 min twice. The coverslips were washed with distilled water extensively, and then stored in distilled water before use.


5


Page 6 of 27

The FAM-labelled polyA-DNA-AuNPs was deposited on the glass slide for 5 min. For the walking device, 5 nM polyA-DNA-AuNPs nanoconjugates were added to 25 mM HEPES buffer solution (pH 7.2, 10 mM MgCl2, and 100 mM NaCl) containing 25 nM DNAzyme walking strand, and incubated for different times at room temperature. The fluorescently nanoparticlebased 3D tarcks were imaged by the TIRF microscopy. All TIRF images were analyzed by a publicdomain image processing software Image J. Each fluorescent bright spot in the TIRF image represents a single particle. The fluorescence of single particle was obtained by measuring the fluorescence intensity of 50 individual bright spots (Average signal intensity = [(1×1 square pixel of 50 individual Exo) - (1×1 square pixel of 50 background area on the image)]/50). Cleavage Efficiency of polyA-DNA-AuNPs. The cleavage efficiency refers to the ratio of the cleaved DNA substrates by DNAzymes to total DNA substrate molecules assembled on AuNPs surface. 5 nM of DNAzyme walking strand and 5 nM FAM-labelled polyA-DNA-AuNPs was mixed in a 25 mM HEPES buffer solution (pH 7.2, 10 mM MgCl2, and 100 mM NaCl), and then 50 µM target adenosine molecules were added and reacted for 5 h. The released FAM-DNA in supernatant was collected by centrifugation. Quantity of FAM-DNA was determined by fluorescence spectroscopy. Excitation and emission wavelengths were set at 488 nm and 526 nm, respectively. All samples were measured in triplicate. In contrast, the cleavage efficiency of thiol-DNA-AuNPs nanoconjugate was also investigated by the same process. The loading value of thiol-DNA on AuNPs surface is ~30 strands per particle, which is consistent with the quantity of polyA20-DNA on single AuNPs surface. Target Detection. Three target molecules, including adenosine, Ag+ and target DNA sequence, were detected by the DNAzyme walker-based sensor. For detection of adenosine and target DNA, 25 mM HEPES buffer (pH 7.2, 10 mM MgCl2, and 100 mM NaCl) was used. For


6

Page 7 of 27 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


the Ag+ detection, 10 mM Tris-HNO3 buffer solution (pH 7.4, 10 mM Mg(NO3)2, 50 mM NaNO3) was prepared. In these testing experiments, the working solution included 5 nM DNAzyme strand and 5 nM FAM-labelled polyA5-DNA-AuNPs. Different concentrations of targets were added to this working solution and reacted for 30 min. The fluorescence of supernatant was measured. Furthermore, the selectivities of this DNAzyme-based walking nanodevice for different targets were investigated respectively. To show the universal property of the DNAzyme walker-based sensor, Hg2+ and microRNA-21 were also selected as targets. FAM-labelled polyA5-DNA-AuNPs (5 nM) nanoconjugates were added to 10 mM Tris-HNO3 buffer (pH 7.4, 10 mM Mg(NO3)2, 50 mM NaNO3) containing 5 nM DNAzyme walking strand and 5 nM target (Hg2+ or target miR-21), and incubated for 20 min.

RESULTS AND DISCUSSION Design of the Target-Initiated 3D DNAzyme Walking Device. The operating principle of the DNAzyme walking device is illustrated in Scheme 1 and Scheme S1. DNA sequences used here are listed in Table S1. This device consists of a spatially isolated 3D track and a targetpowered DNA walker: The 3D track is built on 18-nm sized AuNPs surface that is coated with polyA-tagged DNA substrates containing a ribonucleobase (rA) cleavage point. The walker is a metal-dependent DNAzyme,40,41 which can cleave specifically the DNA substrate track with the assist of Mg2+ ion. The DNAzyme walker contains a catalytic center, two walking arms, and a programmable recognition region. The two arms are designed to be the asymmetrical structure (one is 7 bases long and the other is 15 bases long) that can hybridize with the DNA substrates via Watson-Crick base-pairing. The recognition region is rationally designed for the target-


7


Page 8 of 27

activatable DNAzyme that is inactive in the absence of target and is activated by introduction of various targets such as adenosine, Ag+ ions and target DNA (Scheme S1).

Scheme 1. Schematic for target-initiated stochastic DNAzyme walker that moves processively along the spatially isolated 3D DNA substrate track on AuNPs surface.

Such a DNAzyme-based nanodevice moves autonomously upon the target-initiated cleavage of DNA substrates using a burnt-bridge mechanism. In brief, targets bind specifically to the designed recognition region and trigger the catalytic activity of DNAzyme which cleaves efficiently substrate tracks with Mg2+ cofactor. The short fragment dissociates from the DNAzyme and the long fragment, in contrast, hybridizes stably with the DNAzyme. The unpaired, free short arm of DNAzyme searches for and binds to an adjacent substrate molecule


8

Page 9 of 27 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


on the 3D track. Then, a strand replacement takes place through branch migration, whereby the intact substrate molecule replaces the cleaved substrate fragment to form a more stable, pseudocontinuous DNA duplex. In this process, DNAzyme walker moves from a substrate to a substrate molecule adjacent to it. The repeated movement of DNAzyme walker on a spatially isolated 3D track provides a reproducible way to improve the cleavage efficiency and amplify the biorecognition signals. Single-Particle Fluorescence Analysis for the Operation of the Target-Initiated Stochastic DNAzyme Walker. We first prepared the spatially isolated 3D track on AuNPs surface using the thiol-free oligonucleotides containing the polyA block and the DNA substrate block (Scheme S2a). The resulting nanoconjugate (polyA5-DNA-AuNPs) possessed the excellent dispersity and high stability (Figure S1-S3). The movement of DNAzyme walker initiated by target adenosine on the spatially isolated 3D track on AuNPs surface was then investigated. Carboxyfluorescein (FAM) labelled at the 3’-end of substrate block was employed as the signal reporter. Inactive DNAzyme walker was hybridized onto the 3D substrate track. Target-activated DNAzyme-mediated cleavage of the FAM-labelled substrate resulted in the release of FAM from AuNPs surface and the decreased fluorescence of nanodevices (Figure 1a).


9


Page 10 of 27

Figure 1. Single-particle fluorescence analysis of target-initiated stochastic DNAzyme walker by TIRF microscopy. (a) Schematics for the mechanism of DNAzyme walker moving on the AuNPs surface. (b) TIRF images for DNAzyme walker (FAM-labelled polyA5-DNA-AuNPs) before and after adding adenosine for 30 min. Scale bar = 3 µm. (c) Intensity distribution of nanoparticle labelled with FAM before (red) and after (gray) operation. (d) Representative timelapse images for target-initiated DNAzyme walker moving on a single nanoparticle-based 3D track. (e) Intensity profile for a single nanoparticle-based 3D track over time in the presence (gray) and the absence (red) of target.

We tested the fluorescence change of single nanodevices upon the operation of the DNAzyme walker using total internal reflection fluorescence (TIRF) microscopy.42 In TIRF images, average fluorescent intensity of fluorescent spots decreased significantly after adding target adenosine (1


10

Page 11 of 27 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


µM) (Figure S4). As the operation time went on, the numbers of observed fluorescent particles became less and less. Almost no obvious fluorescent spots could be observed in TIRF images after treatment with adenosine for 30 min (Figure 1b, left panel). Control experiments showed that only a little change in fluorescent intensity and number of bright spots was observed (Figure 1b, right panel) with the addition of adenosine in the absence of DNAzyme walking strands. We also noted that Mg2+ ions play a key role in operating the DNAzyme walker (Figure S5a). Thus, we optimized the concentration of Mg2+ ions, and found that more than 6 mM of Mg2+ ions were suited for the operation of DNAzyme walker (Figure S5b). Statistical analysis of fluorescent intensity of particles (Figure 1c) proved the operating principle of this DNAzyme waking system. A typical walking process was monitored in real time (Figure 1d,e). A rapid decrease in fluorescence intensity was noted in the presence of adenosine, which reached a plateau in 25 min. Further analysis in bulk solution substantiated the target-initiated movement of DNAzyme on AuNPs surface via the burnt-bridge mechanism (Figure S6). Optimization of the PolyA Block Length. Next, we varied the length of polyA block (A5, A10, A15, A20, A25) to precisely spatially control the assembly of DNA substrate blocks on AuNPs surface (Figure 2a), and then quantified the surface density of assembled DNA by a fluorescence method based on the strand displacement (Figure S7).35 The surface density reduced along with the increasing of the length of polyA block (Figure 2b), implying the increasing of interstrand spacing on AuNPs surface. The decrease ratio in density was almost consistent with the increase ratio in the polyA block length (Table S2), indicating that all A bases in the polyA block were completely assembled on AuNPs to achieve full surface coverage. Dynamic light scattering (DLS) measurements were then performed for estimating the conformational changes of the assembled DNA substrates track (Table S2). AuNPs with


11


Page 12 of 27

different polyA blocks (A5, A10, A15, A20, A25) showed uniform hydrodynamic diameters of 33.1-35.2 nm that were independent of the polyA block length. The polyA block changed the surface density rather than the thickness of the assembled layer, thereby indicating that polyADNA on AuNPs surface had an extended and upright conformation that favored hybridization. Subsequently, we further characterized the hybridization ability of the assembled DNA substrates with various polyA block lengths. We found that the hybridization ability was greatly improved with the increase of the length of polyA block (Table S2). With tunable hybridization ability of the assembled substrates, we systematically investigated the catalytic cleavage ability of DNAzyme walkers to substrate blocks on AuNPs. We found that the cleavage efficiency was markedly improved with the increase of the length of polyA block, and reached a maximum (∼92%) with polyA20 blocks (Figure 2c). These results indicated that the enhanced hybridization ability resulting from the reduced density and increased interstrand spacing effectively promoted the cleavage of assembled DNA substrate block. The decreased cleavage efficiency of polyA25-DNA-AuNPs is probably because the increased spacing of substrate blocks limits the movement of DNAzyme walkers.


12

Page 13 of 27 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. Cleavage activity of DNAzyme walker can be regulated by operating the orientation and density of DNA track on AuNP surface. (a) Schematic for spatial control on AuNPs by varying the length of polyA blocks. (b) Surface densities of assembled polyA-DNA (strand/AuNP). (c) Catalytic cleavage efficiency of polyA-DNA on AuNPs by DNAzyme walker in the presence of target.

As a comparison, the classic, thiolated DNA substrate without the polyA block was used to prepare the thiol-DNA-AuNPs. The surface density of thiol-DNA on AuNPs was calculated to be 32±5 molecules per particle, which was roughly equivalent to that of polyA20-DNA. Interestingly, the cleavage efficiency of thiol-DNA substrate was only ∼21%. The finding that


13


Page 14 of 27

the DNAzyme had a high catalytic activity and dynamics towards polyA20-DNA-AuNPs as compared to thiol-DNA-AuNPs and free DNA substrates indicated an improved efficiency and processivity of DNAzyme walker for the spatially isolated 3D track (Figure 3a). Furthermore, we monitored the signalling kinetics of polyA20-DNA-AuNPs with DNAzyme loading (Figure S8). We observed a decreasing dynamics of the DNAzyme nanomachine with the reduction of the DNAzyme-to-substrate ratios from 1:1 to 1:8, and obtained almost the same final fluorescence intensity, which further confirmed the processive movement of DNAzyme walker on the AuNPs-based 3D DNA track.

Figure 3. Cleavage activity of DNAzyme walker on DNA substrate track on AuNP surface. (a) Fluorescence analysis of different nanoparticles and free DNA substrates with the DNAzyme in the presence of target. (b) The conformational effect of 3D track on the catalytic activity of DNAzyme walker.

To further confirm the conformational effect of substrates (3D track) on the efficiency and processivity of this stochastic DNAzyme walker, we employed mercaptohexanol (MCH) to alter the conformation of thiol-based 3D DNA track by passivating the surface of thiol-DNA-


14

Page 15 of 27 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


AuNPs.26,35 The thiol-DNA-AuNPs had a smaller hydrodynamic diameter (26.5 nm) than the polyA20-DNA-AuNPs (Table S2), suggesting that the substrate strands on the thiol-DNAAuNPs surface tended to adopt a “lying-down” conformation that impeded hybridization and cleavage with the DNAzyme. A treatment with MCH increased the hydrodynamic diameter (31.7 nm) of thiol-DNA-AuNPs, indicating a change of “lying-down” conformation to “standing-up” orientation (Scheme S2b). We also found that the DNAzyme activity and cleavage efficiency (∼58%) of thiol-DNA-AuNPs displayed a marked increase with the addition of MCH (Figure 3b). These results proved that the improved catalytic activity of DNAzyme toward polyA-DNAAuNPs is related with conformation of surface-tethered DNA substrates, and indicated that the polyA strategy is more controllable and convenient than the combination of Au-S chemistry and MCH, which probably cause uncertainty in displacement and contamination.31 3D DNAzyme Walking Device for Sensing. The target-initiated DNAzyme movement on the spatially isolated 3D track can result in cascade signal amplification that can be applied for highly sensitive bioanalysis. By simply varying the recognition region of DNAzyme walker, we designed here the versatile DNAzyme walking nanodevices for the detection of different targets (Figure 4a and Scheme S1). In brief, we introduced the aptamer sequence of adenosine to DNAzyme structure for specific detection of adenosine,43 encoded cytosine-rich sequence in the recognition region of DNAzyme walker for selective Ag+ sensing on the basis of cytosine-Ag+cytosine (C-Ag+-C) coordination chemistry,44 and employed a split recognition sequence for detecting target DNA by the complementary base pairing.40,45 The special binding of target and the recognition region of DNAzyme walker triggered the intramolecular configuration change of inactive DNAzyme, and activated its catalytic activity for the cleavage of DNA substrate strands assembled on AuNPs (Scheme S1). After that, the DNAzyme walker moved autonomously from


15


Page 16 of 27

the cleaved substrate fragment to the adjacent intact DNA substrate by a strand replacement and triggered new cleavage (Scheme 1). The process was repeated such that the DNAzyme walker moved continuously on 3D track and produced the enhanced fluorescence signals in supernatant solution for target detection. We first explored the effect of polyA block length on the signal gain to optimize the target analysis. As shown in Figure S9, the signal gain changed from 893% to 1735% with the decrease of the polyA block length. We reasoned that the high signal increase for short polyA block possibly arose from the greater surface density of the DNA substrates on AuNPs surface. Therefore, the 3D track with polyA5 block was used to investigate the sensitivity and selectivity of this DNAzyme walking device.

Figure 4. DNAzyme walking system for target detection. (a) Detection targets can be programmed by designing the recognition region of DNAzyme walker. Relationships between the signal-to-background (F/F0) ratio and concentration of targets, including adenosine (b), Ag+ (c), and target DNA (d), respectively.


16

Page 17 of 27 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


We obtained the dose-response curves for the detection of adenosine, Ag+ and target DNA, respectively (Figure 4b-d). This DNAzyme walker-based sensor showed the detection limit of 10 nM for adenosine, 0.1 nM for Ag+, and 0.1 fM for target DNA sequence (Figure S10). A significant difference among the detection of limit for three targets was noted. This difference was mainly attributed to the different binding affinities of target and its recognition region.40,43-45 It is worth noting that the sensitivity of this sensor show great improvement compared with the reported DNAzymes (Table S3). We also found that the sensor possessed the high selectivity for various targets (Figure S11-S13). Based on our design, other target molecules that can induce the conformational change of DNAzyme can activate the DNAzyme walker.46,47 Similar to Ag+, we introduced the thymine-rich sequence into the recognition region for sensing Hg2+ ions on the basis of thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry.48,49 We also employed the complementary sequence of microRNA-21 (miR-21) as model target miRNA to replace that of target DNA for sensing miR-21. Experimental results showed that both Hg2+ and miR-21 can effectively activate the DNAzyme and drive the operation of DNAzyme walking devices (Figure S14). In addition, the size effect of the AuNPs on the detection limit was investigated. We prepared 13-nm and 30-nm-diameter colloidal AuNPs according to the previously reported literatures.38,50 Then the prepared AuNPs were modified with FAM-labelled polyA5-DNA. Surface densities of assembled polyA5-DNA (strand/AuNP) were determined to be about 67 for 13-nm AuNPs and 292 for 30-nm AuNPs. Finally, we employed the two nanoconjugates to detect adenosine, and obtained their dose-response curves for the detection of adenosine, respectively (Figure S15). The detection limits were calculated to be 60 nM for 13-nm AuNPs and 0.3 nM for 30-nm AuNPs, indicating that the detection sensitivity increased slightly with the increase in the size of


17


Page 18 of 27

the AuNPs. This is mainly because the larger AuNPs can assemble more polyA-DNA molecules and generate stronger fluorescence by the target-induced movement of DNAzyme walker on AuNPs surface. Therefore, the programmable target-initiated DNAzyme walker with improved efficiency and processivity is able to be applied as an universal and desired nanosensor for detecting target molecules with high sensitivity and selectivity.

CONCLUSIONS In summary, we have developed a target-initiated DNAzyme walking device that consists of a target-activatable DNAzyme walker and a spatially isolated 3D track formed by the polyAmediated adsorption of substrates on AuNPs surface. Since polyA block not only provides the anchoring function but also effectively avoids the nonspecific DNA-Au binding, the appended substrate block adopts an upright conformation that facilitates hybridization and cleavage with DNAzyme. The rationally designed DNAzyme activated by target continuously extracts chemical energy from the spatially isolated substrate blocks and uses this energy to effectively drive the operation of the DNAzyme walker. The DNAzyme activity and cleavage efficiency can be improved by simply adjusting the polyA block length, thus indicating that the introduction of polyA provides a better method than the Au-S chemistry to prepare the spatially isolated 3D track with controllable surface density and tunable hybridization efficiency. Our results revealed that the DNAzyme walker-based sensor exhibits the cascade signal amplification for selective and highly sensitive detection of adenosine, Ag+ and target DNA. Thus, the design of targetinitiated DNAzyme walking nanodevice in a reproducible and programmable way holds great promise for developing the high-performance bioinspired DNA nanomachine.


18

Page 19 of 27 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


ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Oligonucleotide sequences; UV-vis spectra; TEM image; fluorescence spectra; TIRF images; kinetics curves; linear relationship; and selectivity data (PDF). AUTHOR INFORMATION Corresponding Author * E-mail (K. Yang): [email protected]. * E-mail (D. He): [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the Hunan Key Laboratory Cultivation Base of the Research and Development of Novel Pharmaceutical Preparations (2016TP1029), National Natural Science Foundation of China (21775036, 31741049), the Hunan Provincial Natural Science Foundation (2018JJ2033, 2018JJ2463), the Research Foundation of Education Bureau of Hunan Province (16A024), and Application Characteristic Discipline of Hunan Province. REFERENCES (1) Schliwa, M.;Woehlke, G. Molecular Motors. Nature 2003, 422, 759-765.


19


Page 20 of 27

(2) Kay, E. R.; Leigh, D. A.; Zerbetto, F. Synthetic Molecular Motors and Mechanical Machines. Angew. Chem. Int. Ed. 2007, 46, 72-191. (3) Hirokawa, N.; Noda, Y.; Tanaka, Y.; Niwa, S. Kinesin Superfamily Motor Proteins and Intracellular Transport. Nat. Rev. Mol. Cell Biol. 2009, 10, 682-696. (4) Yin, P.; Choi, H. M. T.; Calvert, C. R.; Pierce, N. A. Programming Biomolecular SelfAssembly Pathways. Nature 2008, 451, 318-322. (5) Zhou, C.; Duan X.; Liu, N. A Plasmonic Nanorod that Walks on DNA Origami. Nat. Commun. 2015, 6, 8102. (6) Tian, Y.; He, Y.; Chen, Y.; Yin, P.; Mao, C. Molecular Devices-a DNAzyme that Walks Processively and Autonomously along a One-Dimensional Track. Angew. Chem. Int. Ed. 2005, 44, 4355-4358. (7) Yehl, K.; Mugler, A.; Vivek, S.; Liu, Y.; Zhang, Y.; Fan, M.; Weeks E. R.; Salaita, K. High-Speed DNA-Based Rolling Motors Powered by RNase H. Nat. Nanotechnol. 2016, 11, 184-190. (8) Omabegho, T.; Sha, R.; Seeman, N. C. A Bipedal DNA Brownian Motor with Coordinated Legs. Science 2009, 324, 67-71. (9) Bath, J.; Green S. J.; Turberfield, A. J. A Free-Running DNA Motor Powered by a Nicking Enzyme. Angew. Chem., Int. Ed. 2005, 44, 4358-4361.


20

Page 21 of 27 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


(10) You, M.; Chen, Y.; Zhang, X.; Liu, H.; Wang, R.; Wang, K.; Williams, K. R.; Tan, W. An Autonomous and Controllable Light-Driven DNA Walking Device. Angew. Chem., Int. Ed. 2012, 51, 2457-2460. (11) Pei, R.; Taylor, S. K.; Stefanovic, D.; Rudchenko, S.; Mitchell, T. E.; Stojanovic, M. N. Behavior of Polycatalytic Assemblies in a Substrate-Displaying Matrix. J. Am. Chem. Soc. 2006, 128, 12693-12699. (12) Yang, Y.; Goetzfried, M. A.; Hidaka, K.; You, M.; Tan, W.; Sugiyama, H.; Endo, M. Direct Visualization of Walking Motions of Photocontrolled Nanomachine on the DNA Nanostructure. Nano Lett. 2015, 15, 6672-6676. (13) Wickham, S. F. J.; Bath, J.; Katsuda, Y.; Endo, M.; Hidaka, K.; Sugiyama, H.; Turberfield, A. J. A DNA-Based Molecular Motor that can Navigate a Network of Tracks. Nat. Nanotechnol. 2012, 7, 169-173. (14) Lund, K.; Manzo, A. J.; Dabby, N.; Michelotti, N.; Johnson-Buck, A.; Nangreave, J.; Taylor, S.; Pei, R.; Stojanovic, M. N.; Walter, N.; Winfree, E.; Yan, H. Molecular Robots Guided by Prescriptive Landscapes. Nature 2010, 465, 206-209. (15) Yang, X. L.; Tang, Y. A.; Mason, S. D.; Chen J. B.; Li, F. Enzyme-Powered ThreeDimensional DNA Nanomachine for DNA Walking, Payload Release, and Biosensing. ACS Nano 2016, 10, 2324-2330. (16) Hwang, M. T.; Landon, P. B.; Lee, J.; Choi, D.; Mo, A. H.; Glinsky, G.; Lal, R. Highly Specific SNP Detection using 2D Graphene Electronics and DNA Strand Displacement. Proc. Natl. Acad. Sci. USA 2016, 113, 7088-7093.


21


Page 22 of 27

(17) Yuan, L.; Giovanni, M.; Xie, J. P.; Fan, C. H.; Leong, D. T. Ultrasensitive IgG Quantification using DNA Nano-pyramids. NPG Asia Mater. 2014, 6, e112. (18) Cha, T.-G.; Pan, J.; Chen, H.; Salgado, J.; Li, X.; Mao, C.; Choi, J. H. A Synthetic DNA Motor that Transports Nanoparticles along Carbon Nanotubes. Nat. Nanotechnol. 2014, 9, 39-43. (19) Wickham, S. F. J.; Endo, M.; Katsuda, Y.; Hidaka, K.; Bath, J.; Sugiyama, H.; Turberfield, A. J. Directing Self-Assembly of DNA Nanotubes using Programmable Seeds. Nat. Nanotechnol. 2011, 6, 166-169. (20) He, Y.; Liu, D. R. Autonomous Multistep Organic Synthesis in a Single Isothermal Solution Mediated by a DNA Walker. Nat. Nanotechnol. 2010, 5, 778-782. (21) Meng, W.; Muscat, R. A.; McKee, M. L.; Milnes, P. J.; El-Sagheer, A. H.; Bath, J.; Davis, B. G.; Brown, T.; O’Reilly, R. K.; Turberfield, A. J. An Autonomous Molecular Assembler for Programmable Chemical Synthesis. Nat. Chem. 2016, 8, 542-548. (22) Liu, X. Q.; Lu, C. H.; Willner, I. Switchable Reconfiguration of Nucleic Acid Nanostructures by Stimuli-Responsive DNA Machines. Acc. Chem. Res. 2014, 47, 1673-1680. (23) Jung, C.; Allen, P. B.; Ellington, A. D. A Stochastic DNA Walker that Traverses a Microparticle Surface. Nat. Nanotechnol. 2016, 11, 157-163. (24) Zhang, H.; Lai, M.; Zuehlke, A.; Peng, H.; Li, X.-F.; Le, X. C. Binding-Induced DNA Nano machines Triggered by Proteins and Nucleic Acids. Angew. Chem. Int. Ed. 2015, 54, 14326-14330.


22

Page 23 of 27 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


(25) Taghdisi, S. M.; Danesh, N. M.; Ramezani, M.; Emrani, A. S.; Abnous, K. Novel Colorimetric Aptasensor for Zearalenone Detection Based on Nontarget-Induced Aptamer Walker, Gold Nanoparticles, and Exonuclease-Assisted Recycling Amplification. ACS Appl. Mater. Interfaces 2018, 10, 12504-12509. (26) Qu, X.; Zhu, D.; Yao, G.; Su, S.; Chao, J.; Liu, H.; Zuo, X.; Wang, L.; Shi, J.; Wang, L.; Huang, W.; Pei, H.; Fan, C. An Exonuclease III-Powered, On-Particle Stochastic DNA Walker. Angew. Chem. Int. Ed. 2017, 56, 1855-1858. (27) Peng, H.; Li, X.-F.; Zhang, H.; Le, X. C. A MicroRNA-Initiated DNAzyme Motor Operating in Living Cells Nat. Commun. 2017, 8, 14378. (28) Liang, C.; Ma, P.; Liu, H.; Guo, X.; Yin, B.; Ye, B. Rational Engineering of Dynamic, Entropy-Driven DNA Nanomachine for Intracellular MicroRNA Imaging. Angew. Chem. Int. Ed. 2017, 56, 9077-9081. (29) Ma, P.; Liang, C.; Zhang, H.; Yin, B.; Ye, B. A Highly Integrated DNA Nanomachine Operating in Living Cells Powered by an Endogenous Stimulus. Chem. Sci. 2018, 9, 3299-3304. (30) Parak, W. J.; Pellegrino, T.; Micheel, C. M.; Gerion, D.; Williams, S. C.; Alivisatos, A. P. Conformation of Oligonucleotides Attached to Gold Nanocrystals Probed by Gel Electrophoresis. Nano Lett. 2003, 3, 33-36. (31) Park, S.; Brown K. A.; Hamad-Schifferli, K. Changes in Oligonucleotide Conformation on Nanoparticle Surfaces by Modification with Mercaptohexanol. Nano Lett. 2004, 4, 1925-1929.


23


Page 24 of 27

(32) Schreiner, S. M.; Shudy, D. F.; Hatch, A. L.; Opdahl, A.; Whitman, L. J.; Petrovykh, D. Y. Controlled and Efficient Hybridization Achieved with DNA Probes Immobilized Solely Through Preferential DNA-Substrate Interactions. Anal. Chem. 2010, 82, 2803-2810. (33) Opdahl, A.; Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Independent Control of Grafting Density and Conformation of Single-Stranded DNA Brushes. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9-14. (34) Chen L.; Chao, J.; Qu, X.; Zhang, H.; Zhu, D.; Su, S.; Aldalbahi, A.; Wang, L.; Pei, H. Probing Cellular Molecules with PolyA-Based Engineered Aptamer Nanobeacon. ACS Appl. Mater. Interfaces 2017, 9, 8014-8020. (35) Pei, H.; Li, F.; Wan, Y.; Wei, M.; Liu, H.; Su, Y.; Chen, N.; Huang, Q.; Fan, C. Designed Diblock Oligonucleotide for the Synthesis of Spatially Isolated and Highly Hybridizable Functionalization of DNA-Gold Nanoparticle Nanoconjugates. J. Am. Chem. Soc. 2012, 134, 11876-11879. (36) Yao, G.; Pei, H.; Li, J.; Zhao, Y.; Zhu, D.; Zhang, Y.; Lin, Y.; Huang Q.; Fan, C. Clicking DNA to Gold Nanoparticles: Poly-Adenine-Mediated Formation of Monovalent DNA-Gold Nanoparticle Conjugates with Nearly Quantitative Yield. NPG Asia Mater. 2015, 7, e159. (37) Zhu, D.; Song, P.; Shen, J.; Su, S.; Chao, J.; Aldalbahi, A.; Zhou, Z.; Song, S.; Fan, C.; Zuo, X.; Tian, Y.; Wang, L.; Pei, H. PolyA-Mediated DNA Assembly on Gold Nanoparticles for Thermodynamically Favorable and Rapid Hybridization Analysis. Anal. Chem. 2016, 88, 49494954.


24

Page 25 of 27 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


(38) Vetten, M. A.; Tlotleng, N.; Rascher, D. T.; Skepu, A.; Keter, F. K.; Boodhia, K.; Koekemoer, L. A.; Andraos, C.; Tshikhudo, R.; Gulumian, M. Label-Free in Vitro Toxicity and Uptake Assessment of Citrate Stabilised Gold Nanoparticles in Three Cell Lines. Part. Fibre Toxicol. 2013, 10, 50. (39) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. A Fluorescence-Based Method for Determining the Surface Coverage and Hybridization Efficiency of Thiol-Capped Oligonucleotides Bound to Gold Thin Films and Nanoparticles. Anal. Chem. 2000, 72, 5535-5541. (40) He, D.; He, X.; Yang X.; Li, H.-W. A Smart ZnO@polydopamine-Nucleic Acid NanoSystem for Ultrasensitive Live Cell mRNA Imaging by the Target-Triggered Intracellular SelfAssembly of Active DNAzyme Nanostructures. Chem. Sci. 2017, 8, 2832-2840. (41) Wang, F.; Elbaz, J.; Orbach, R.; Magen, N.; Willner, I. Amplified Analysis of DNA by the Autonomous Assembly of Polymers Consisting of DNAzyme Wires. J. Am. Chem. Soc. 2011, 133, 17149-17151. (42) Liang, L.; Li, J.; Li, Q.; Huang, Q.; Shi, J. Y.; Yan, H.; Fan, C. H. Single-Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells. Angew. Chem. Int. Ed. 2014, 53, 7745-7750. (43) Song, P.; Xiang, Y.; Xing, H.; Zhou, Z.; Tong, A.; Lu, Y. Label-Free Catalytic and Molecular Beacon Containing an Abasic Site for Sensitive Fluorescent Detection of Small Inorganic and Organic Molecules. Anal. Chem. 2012, 84, 2916-2922.


25


Page 26 of 27

(44) Freage, L.; Wang, F.; Orbach, R.; Willner, I. Multiplexed Analysis of Genes and of Metal Ions Using Enzyme/DNAzyme Amplification Machineries. Anal. Chem. 2014, 86, 11326-11333. (45) Zhou, W.; Chen, Q.; Huang, P.-J. J.; Ding, J.; Liu, J. DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum. Anal. Chem. 2015, 87, 4001-4007. (46) Lai, W.; Ren, L.; Tang, Q.; Qu, X.; Li, J.; Wang, L.; Li, L.; Fan, C.; Pei, H. Programming Chemical Reaction Networks Using Intramolecular Conformational Motions of DNA. ACS Nano 2018, 12, 7093-7099. (47) Qu, X.; Wang, S.; Ge, Z.; Wang, J.; Yao, G.; Li, J.; Zuo, X.; Shi, J.; Song, S.; Wang, L.; Li, L.; Pei, H.; Fan, C. Programming Cell Adhesion for On-Chip Sequential Boolean Logic Functions. J. Am. Chem. Soc. 2017, 139, 10176-10179. (48) Qi, L.; Xiao, M.; Wang, F.; Wang, L.; Ji, W.; Man, T.; Aldalbahi, A.; Khan, M. N.; Periyasami, G.; Rahaman, M.; Alrohaili, A.; Qu, X.; Pei, H.; Wang, C.; Li, L. Poly-CytosineMediated Nanotags for SERS Detection of Hg2+. Nanoscale 2017, 9, 14184-14191. (49) Freeman, R.; Finder, T.; Willner, I. Multiplexed Analysis of Hg2+ and Ag+ Ions by Nucleic Acid Functionalized CdSe/ZnS Quantum Dots and Their Use for Logic Gate Operations. Angew. Chem. Int. Ed. 2009, 48, 7818-7821. (50) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Determining the Size and Shape Dependence of Gold Nanoparticle Uptake into Mammalian Cells. Nano Lett 2006, 6, 662-668.


26

Page 27 of 27 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 content


27

A Programmable Target-Initiated DNAzyme Walker Walking along a

Recommend Documents