Exploration of the Kinetics of Toehold-Mediated Strand Displacement

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Exploration of the Kinetics of ToeholdMediated Strand Displacement via Plasmon Rulers Mei-Xing Li,† Cong-Hui Xu,† Nan Zhang, Guang-Sheng Qian, Wei Zhao,* Jing-Juan Xu,* and Hong-Yuan Chen 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 210023, China S Supporting Information *

ABSTRACT: DNA/RNA strand displacement is one of the most fundamental reactions in DNA and RNA circuits and nanomachines. In this work, we reported an exploration of the dynamic process of the toehold-mediated strand displacement via core−satellite plasmon rulers at the single-molecule level. Applying plasmon rulers with unlimited lifetime, single-strand displacement triggered by the invader that resulted in stepwise leaving of satellite from the core was continuously monitored by changes of scattering signal for hours. The kinetics of strand displacement in vitro with three different toehold lengths have been investigated. Also, the study revealed the difference in the kinetics of strand displacement between DNA/RNA and DNA/DNA duplexes. For the kinetics study in vivo, influence from the surrounding medium has been evaluated using both phosphate buffer and cell lysate. Applying core−satellite plasmon rulers with high signal/noise ratio, kinetics study in living cells proceeded for the first time, which was not possible by conventional methods with a fluorescent reporter. The plasmon rulers, which are flexible, easily constructed, and robust, have proven to be effective tools in exploring the dynamical behaviors of biochemical reactions in vivo. KEYWORDS: plasmon rulers, dark-field microscopy, strand displacement, kinetics, single-molecule imaging displacement.1,2,18−20 The typical fluorescence studies greatly improved the understanding of the kinetics of nucleic acid reactions and helped the design of dynamic DNA and RNA circuits and nanomachines. Nevertheless, the limitations of fluorescent dyes such as photobleaching and blinking restricted the study in vivo. In addition, using a fluorescent reporter system, an additional reporting reaction was required, which made the system more complicated. Another optical characterization relied on plasmonic resonance of noble metal nanomaterials developed fast in the past decades. The scattering wavelength of plasmonic nanoparticles is highly sensitive to the shape, structure, surrounding environment, and distance between the particles,21−24 which facilitates the surface engineering and single-molecule assay.25 In addition, the plasmonic probes without photobleaching or blinking allow continuous imaging for hours.26 By recording the wavelength shifts or intensity changes of the plasmonic probes, some important biological processes, such as DNA bending and

DNA nanotechnology, broadly divided into structural and dynamic DNA nanotechnology, has been applied in constructing DNA self-assembly objects with complexity and engineering reconfigurable and autonomous devices.1 DNA strand displacement, as one of the most fundamental and useful reactions in dynamic DNA nanodevices, is usually initiated by a short sequence of single-strand domain names as the toehold and causes the displacement of one strand of DNA of another in binding to a third strand with partial complementarity to both.2 Such enzyme-free strand displacement that has been investigated since 1970s and boomed in the following years was widely employed in the areas of dynamic DNA technology as a basic mechanism especially in operating biological logic circuits,3−6 noncovalent DNA catalysis,7−9 autonomous DNA nanomachines,10−14 as well as reconfiguration of DNA nanostructures15−18 at the nanoscale. The kinetics of toehold-mediated strand displacement are strongly dependent on the length and sequence of the toehold.2 Some impressive pioneering works have been reported in revealing the control of DNA strand displacement using a toehold, and it was possible to predict the kinetics of different strand displacement reactions and impact of mismatches on © XXXX American Chemical Society

Received: December 7, 2017 Accepted: March 26, 2018 Published: March 26, 2018 A

DOI: 10.1021/acsnano.7b08673 ACS Nano XXXX, XXX, XXX−XXX

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ACS Nano Scheme 1. Schematic Illustration of Experimental Design and Process of Toehold-Mediated Strand Displacement

complementary to the β̅ domain of a single-strand DNA (sDNA) on satellite GNPs. From UV−vis absorbance intensities (Figure S2), fluorescence experiment (Figure S3), and theoretical calculation (Figure S4), each satellite GNP was probably bonded to the core through a single DNA duplex. A detailed determination process is shown in the Supporting Information. According to dynamic light scattering measurements, hydrodynamic diameters of satellite GNP, core GNP, and core−satellite nanostructure were around 16.5, 51.5, and 85.7 nm, respectively (Figure S1d). The structure of the core− satellite nanoassembly was further characterized using highresolution transmission electron microscopy (Figure S5a). The number of satellites around the core was statistically obtained from TEM images by calculating 50 nanoassemblies (Figure S5b). As shown in Figure S5, the satellite number ranges from 8 to 12, mainly at 10 and 11 (75%). However, it was hard to precisely estimate the distance between core and satellite GNPs from these TEM images because the 3D core−satellite structure was rearranged to a 2D-packed structure during the drying process.40 To make an accurate estimation, we applied GNPs with equal size (50 nm in diameter) and synthesized the GNP−dimer using the same ssDNA sequences (cDNA and sDNA) as used for the core−satellite nanoassembly. With two GNPs of the dimer lying in the same plane on the TEM grid, the space between them should be closer to the real condition in the experiments. After statistical analysis of the GNP−dimers from TEM images, the interparticle distance (0.7−1.5 nm) was statistically analyzed and averaged at 1.2 nm (Figure S6). The plasmon-coupling-induced scattering variation was investigated using DFM and spectrometry. As shown in Figure 1, core GNPs modified with cDNA were observed as dark green spots with the scattering wavelength centered at 560 nm. The modification of DNA strands changed the dielectric layer around the nanoparticle,41 resulting in a slight red shift and change in the peak shape (Figure S7). The core−satellite assemblies scattered bright salmon pink spots with λmax of 622 nm. Compared with the core GNP, the core−satellite nanoassembly exhibited highly increased scattering intensity (ca. 10 times) and a significant red shift (Δλ = 62 nm), which made such a probe an excellent plasmon ruler for the study of dynamic processes. From theoretical simulation using the finitedifference time-domain (FDTD), Δλ of 68 nm was estimated in comparison to that of the core GNP (λmax = 544 nm) with 12 satellite assemblies (λmax = 612 nm) (Figure S8), which

cleavage,27 RNA digestion,28 activity of single telomerase,29 and single DNA hybridization,30 have been read out. As the kinetics of strand displacement strongly depend on the matrix, to build models both in vitro and in living cells, “plasmon rulers” could be suitable candidates. Herein, inspired by the continuous imaging of plasmon rulers to measure dynamic biophysical processes,31 we synthesized a core−satellite gold nanoparticle assembly and investigated the kinetics of toehold-mediated strand displacement at the surface of gold nanoparticles (GNPs). The core−satellite nanoassembly was composed of several “satellite” nanoparticles tethered by duplex DNA to a “core” nanoparticle. The strand displacement accompanied by the leaving of the satellite from the core resulted in the blue shift of scattering wavelength and changes of color, which could be monitored under dark-field microscopy (DFM) (Scheme 1a). Without an additional reaction, the kinetics of strand displacement could be studied at the single-molecule level. We chose microRNA-21 (miRNA21), the oncogene, as the invading strand because it was overexpressed in many cancer cells32−39 and allowed us to demonstrate the model in living cells. The studies in vitro revealed the difference in the kinetics of strand displacement between DNA/RNA and DNA/DNA duplexes and helped us learn the relationship between toehold length and the reaction kinetics. Compared with traditional fluorescent dyes, plasmon rulers are more robust and enable continuous observation for hours. With several plasmon rulers, displacement kinetics could be examined both in vitro and in living cells. The proposed method offers an opportunity to study the kinetics in vivo or under extremely low concentration (at single-molecule level), which could not be achieved before. In addition, using reaction constants in cell lysate for calibration, the expression of miRNA-21 in living cells could be easily deduced, which was much simpler than using real-time polymerase chain reaction (RT-PCR).

RESULTS AND DISCUSSION Synthesis of Core−Satellite Nanoparticle Plasmon Rulers. The core−satellite nanostructure was formed with a core GNP (ca. 49 nm in diameter) and small satellite GNPs (ca. 13.8 nm in diameter) (Figure S1) bonded to the core via DNA strand hybridization. As shown in Scheme 1b, the core GNP was functionalized with complementary DNA (cDNA) that comprised α and β domains, in which the β domain was B

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We defined apparent rate constant kobs by multiplying the displacement rate constant k with constant concentration [I].

kobs = k[I]

A detailed rate constant fitting procedure is demonstrated in the Materials and Methods section. In conventional fluorescent measurements, an additional reporter system is needed, in which the displaced strand displaces a fluorescently labeled strand from a reporter duplex.19 Therefore, rate constant of the reporting reaction (krep) had to be input in the mechanism study. Here, in the experiments with plasmon rulers, each single-strand displacement could be monitored for the kinetics study. The cumulative probability (P) of strand displacement represented the degree of conversion, which was calculated by dividing the number of satellite leaving events up to a given time, by the total number of events observed during the experimental time course. The expression of P was deduced as a function of time:

Figure 1. Core−satellite nanoparticle plasmon rulers. The scattering image of separated cDNA modified core GNPs (a) and core−satellite nanoassemblies (b) under DFM; the scale bar of the inset TEM image is 20 nm. The scattering spectra (c) of a single nanoassembly (red) and a single core GNP (black).

fitted well with the experimental results. It is worth noting that, although two orthogonal polarized lights were applied, the simulation model was still greatly simplified compared with experimental conditions. From FDTD simulation with a totalfield scattered-field source, two scattering peaks are shown for the GNP core−satellite nanoassembly. However, the obtained spectrum is composed of a sum of the two peaks, which could not be simply fitted with one Lorentzian line shape. Kinetic Mode of the Strand Displacement Process. The toehold-mediated strand displacement at the surface of the core GNP was described by the three-step model2 (Scheme 1b). The hybridized double strand is composed of sDNA (S) with a toehold domain (γ)̅ and complementary DNA (C). The invading strand, abbreviated as strand I, consisted of β and γ domains, in which the γ domain was the toehold complementary sequence (Table 1). First, invading strand I bonds to

P = 1 − exp( −kobst )

sequences

length (nt)

α β γ

5′-TTT-3′ 5′-TCAGACTGATGTTGA-3′ 5′-TAGCTTA-3′

3 15 7

the exposed toehold γ ̅ and then, after the branch migration, the β domain of complementary DNA was displaced by the invader with the same oligonucleotide sequences. Finally, complementary DNA was released, and a new double strand of I+S was formed. Each strand displacement event made one satellite leave the core and could induce shifting of a scattering wavelength (Figure S8). In other words, the strand displacement process could be monitored via the scattering spectra of plasmon rulers at the single-molecule level for the kinetics study. As previously reported, at sufficiently low concentration, the kinetics of toehold-mediated strand displacement are wellapproximated by biomolecular reactions with second-order rate constants.2 Assuming displacement complexes quickly resolve into products,42 the toehold-mediated strand displacement process could be shown as k

CS + I → C + IS

(3)

The apparent rate constant kobs could be deduced based on the observed P. Also, the second-order constant k could be fitted with a reference value compared to the results from the conventional fluorescence method. Analyses of Plasmon Rulers for Strand Displacement. We first investigated the dynamical strand displacement process under in vitro conditions of 10 mM PBS at room temperature. The core−satellite nanoassemblies were immobilized on the surface of an amino-group-functionalized silane-treated glass, covered with ultrathin cover glasses and sealed by PDMS mode. The scattering colors and intensities were recorded under DFM with serial imaging of all particles in the field-of-view. After we added 5.0 nM invader (miRNA-21), the initial bright salmon pink spots turned yellow and then dark green as time elapsed (Figure 2b). The scattering spectra of the selected signalassembled nanoparticle was also monitored exactly after every DFM image was taken (Figure 2c). The scattering wavelength showed a stepwise blue shift corresponding to the individual strand displacement event (Figure 2d). Every strand displacement event would cause the wavelength to blue shift 5 nm on average. As the scattering intensity and wavelength changed synchronously with the R/G drops, and color analysis was much easier, we chose the stepwise decrease of R/G to indicate the strand displacement events for the kinetics study. As the color changed dramatically, we did color analysis by evaluating a series of images using ImageJ and extracted the red/green (R/G) value of each separate spot. As shown in Figure 3a, after addition of 10 nM invader (miRNA-21), stepwise drops of R/G value with certain waiting time (tw) were observed at one single spot. Meanwhile, the gray value also decreased step by step with time elapsing (Figure S9), indicating the dimming of scattering intensity. In control experiments with addition of an RNA strand mismatched with sDNA (10 nM miRNA-141) (Figure 3a, inset) and without addition of the invader (Figure S10), no obvious drop of R/G was observed, confirming that the stepwise signal changes were from the leaving events of satellite GNPs. In an ideal mode, each step indicated a single-strand displacement event, but in the experiment, two or more strands might be displaced in the same detection time, resulting in a larger height of steps (ΔR/ G). In the statistical study, we took four concentrations (0.5, 1.0, 5.0, and 50.0 nM) of the invader and 40 plasmon rulers in total to evaluate the ΔR/G of 380 steps. According to the

Table 1. Domain Sequences domain

(2)

(1)

where k is the second-order rate constant. In this system, compared to the CS double strand (0.4 pM in 30 μL reservoir), the amount of the invader (0.1−200 nM) was substantially in excess, which remained constant during the whole displacement process. The process appears as a pseudo-first-order reaction. C

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Figure 2. DFM imaging and spectra analyses of plasmon rulers. (a) Strand-displacement-mediated plasmon rulers. (b) Series of DFM images monitored after the addition of the invading strand (scale bar: 5 μm). (c) Scattering spectra of the selected nanoassembly in the strand displacement. (d) Intensity changes (ΔI = I1 − I0, where I0 is the original intensity value and I1 is the intensity at time t1 during the displacement) and wavelength shifts (Δλ = λ1 − λ0, where λ0 is the original peak wavelength and λ1 is the peak wavelength at time t1 during the displacement).

Figure 3. R/G value analyses for strand displacement events. (a) R/G value trace (black dots) and fitted stepwise signal (red line) of a single nanoassembly with time elapsing (inset: R/G value trace of displacement process with miRNA-141 as invader strand). (b) Distribution of R/ G value changes for every step height during the strand displacement process. (c) Statistical analyses of the leaving events of satellite GNPs from the core with different concentrations (0.1−200 nM) of the invader as a function of time.

distribution, 0.045 was defined as the ΔR/G caused by a single event (Figure 3b). The exact number of events up to a given time was calculated by dividing the recorded ΔR/G by 0.045

and rounding the number. The leaving events were recorded after adding different concentrations of the invader. As shown in Figure 3c, along with the increasing concentration of the D

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Figure 4. Rate constant fitting of in vitro strand displacement process. The cumulative probability of strand displacement under different concentrations of (a) miRNA-21 and (c) DNA invader in PBS solution as a function of time. The fitted rate constants of different concentrations for (b) miRNA-21 and (d) DNA invader.

ratio was too large to obtain convincing experimental results. Therefore, finally, the concentrations of both [CS] complex and invader strands were set to 10 nM, and the second-order constant k was fitted to 7.14 × 105 M−1 s−1. Compared with k deduced from a plasmon ruler (1.31 × 105 M−1 s−1), it is 5.5 times larger. The reasons were explained from two aspects. First, the bimolecular reaction model that defines the strand displacement as a second-order process does not account for intermediate concentrations. The initial concentrations of CS and I could influence the accuracy of the BM model. As the real conditions (0.4 pM [CS]) of the proposed method and fluorescence method (10 nM [CS] and [I]) are significantly different, we could imagine some variation of k. Second, the core−satellite system is a heterogeneous system. Although the toehold was designed to locate at the end far from the satellite, the hybridization of DNA/RNA strands might still be affected by the steric effects. In addition, the release of GNP attached onto the surface of the core GNP may not happen simultaneously with the strand displacement. These problems might be solved by designing a flowing system. However, for the comparison of in vitro and in vivo (static environment) kinetics, the obtained kinetics are informative. Also, the proposed method offers an opportunity for the kinetics study under extremely low concentrations (at the single-molecule level), which could not be achieved using a conventional fluorescence method. The kinetics study with three different lengths of toehold (6, 7, and 9 nt) was also performed. As shown in Figure S13, the fitted reaction constants are 8.62 × 104 and 1.38 × 105 M−1 s−1 with 6 and 9 nt, respectively. From the results, it was found that the reaction rate of the toehold-mediated strand displacement reaction saturated for a toehold of 7 nt. However, in contrast to the strand displacement of DNA/RNA, the saturated toehold lengths for DNA/DNA and RNA/RNA strand displacement are noticeable different as 62 and 4 nt,43 respectively. As is wellknown, for short toeholds, the invading strand would fall off from the toehold and undergo frequent binding and unbinding

invader, the distribution of leaving events was more concentrated. It should be noted that the uniformity of scattering response during satellite leaving events could influence the accuracy of the plasmon-ruler-based method. From the FDTD simulations with different structure models, it is hard to define an absolute uniform response because the simulation model was simplified compared with experimental conditions. Therefore, the distributions of R/G value changes (ΔR/G) were assessed during different periods of time. As shown in Figure S11, with 10 nM invader, the averaged ΔR/G in three time periods is 0.049, 0.041, and 0.045. Therefore, the scattering spectra response was approximately equal with different numbers of satellites in the experiments. In Vitro Studies of Strand Displacement Kinetics. Based on the statistical event distribution of 20 plasmon rulers and more than 200 events (Figure 3c), the P value of strand displacements (7 nt toehold length) was calculated and fitted with eq 3 (Figure 4a). The fitting curves yielded kobs (0.61 × 10−4 to 3.98 × 10−4 s−1) at different concentrations of the invader. At sufficiently low concentration of miRNA-21 (100 pM to 2.0 nM), kobs was linear to the concentration (R = 0.992), and k was fitted as 1.31 × 105 M−1 s−1. However, when the concentration of miRNA-21 was larger than 2.0 nM, the linear correlation between kobs and the concentration became worse. Because the concentrations of intermediate products have not been accounted for, the concentrations of [CS] and [I] must be sufficiently low to accurately describe the kinetics of strand displacement processes.2 The results derived using plasmon rulers were compared with those obtained using a conventional fluorescent method. As the length of the α domain in cDNA was ca. 1 nm in the present work, the fluorescence of Cy5 labeled at the 3′ end of cDNA could be quenched by BHQ labeled at the 5′ end of sDNA (Figure S12). The dynamic process was recorded with a fluorescence photometer. At first, we tried to use low concentrations ( DNA/DNA > hybrids, and it is reasonable to conclude that the strand displacement of DNA/ RNA needs longer toehold length as the stability of hybrids is less than those of RNA and DNA duplexes. The melting temperatures (Tm) of the formed DNA/RNA duplex and DNA/DNA duplex under different concentrations in the present work have been calculated and are shown in Figure S14, which confirmed the thermodynamic stability of the duplex is lower than that of DNA/DNA. We also examined DNA/DNA strand displacement with the same sequences as DNA/RNA, where the displacement rate constant k was fitted to be 3.26 × 105 M−1 s−1, which was much higher than that of DNA/RNA (1.31 × 105 M−1 s−1). Fluorescence experiments confirmed the correctness of the conclusion (Figure S12). We tried to explain such a phenomenon from both the secondary structures of DNA and RNA invader strands and the thermodynamic stabilities of formed DNA/DNA and DNA/RNA duplexes. Because the strand displacement requires opening of the stem of the invader, NUPACK software was used for thermodynamic prediction of the secondary structures of RNA and DNA invader strands. As shown in Figure S15, the free energy of RNA was lower than that of the DNA strand with the same sequences, which indicated that more energy would be needed to break the relatively stable secondary structure of microRNA21 in the solution.44 Further, as previously mentioned, the stability of the hybrids is lower than that of the DNA duplex; therefore, it is reasonable to expect a higher reaction rate F

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All the sequences are shown in Table S1. Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·4H2O) and sodium citrate were obtained from Sinopharm Chemical Reagent Co., Ltd. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), (3-aminopropyl)triethoxysilane (APTES, 99%), O-(3-carboxypropyl)-O′-[2-(3-mercaptopropionylamino)ethyl]polyethylene glycol (Mw 3000, SH-PEG-COOH), bis(psulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-Nhydroxysuccinimide ester sodium salt (sulfo-SMCC) were provided by Sigma-Aldrich. All other reagents were of analytic grade and used without further purification. The Millipore (model Milli-Q) ultrapure water (resistivity of 18.2 MΩ·cm) was RNase-free by being pretreated with diethylpyrocarbonate and was used throughout the experiment. The 1× PBS solution used in the cell culture was purchased from Key GEN Biotech, and other buffer solutions used in the experiments were prepared as follows: PBS (10 mM Na2HPO4/NaH2PO4, pH 7.4), TrisHCl buffer (10 mM Tris, pH 7.4). Apparatus. Transmission electron microscopy was performed with a JEM-1011 instrument (JEOL Ltd., Japan). High-resolution transmission electron microscopy was performed with a JEM-2100 transmission electron microscope (JEOL Ltd., Japan). The UV−vis absorption spectra were obtained on a Shimadzu UV-3600 UV−vis− NIR photospectrometer (Shimadzu Co., Japan) at room temperature. The dynamic light scattering data were acquired with a Malvern (Nano-Z, Malvern Instruments Ltd., Britain) instrument. The fluorescence experiments were conducted on fluorescence spectrophotometer (F-7000, Hitachi Ltd., Japan). The dark-field measurements were carried out on an inverted microscopy (IX71, Olympus) that was equipped with a dark-field condenser (0.8 < NA < 0.92) and a 60× objective lens (NA = 0.7). A 100 W halogen lamp was used as the white light source. A true-color digital camera (Olympus DP80, Japan) was used to capture the dark-field color images. The scattering light of the gold nanoparticle was split by a monochromator (Acton SP2358, PI, USA) equipped with a grating (grating density, 300 lines/mm; blazed wavelength, 500 nm) and recorded by an excelon EMCCD (400BR, PI, USA) to obtain the scattering spectra. Preparation of the Satellite Gold Nanoparticles. The satellite gold nanoparticles with an average diameter of 13 nm were prepared by the citrate reduction of HAuCl4 according to a classical method with some modification.45 Briefly, all glassware was thoroughly cleaned in aqua regia (three parts concentrated HCl, one part concentrated HNO3), rinsed with ultrapure water, and oven-dried prior to use. Fifty milliliters of HAuCl4 (0.01%) was added to the cleaned round-bottom flask with vigorous stirring and heated to 100 °C. Then 5 mL of trisodium citrate (38.8 mM) was quickly added. After the color changed from pale yellow to burgundy, the mixture was kept boiling for 15 min. The oil bath was then removed, and stirring was continued for another 15 min. After being cooled to room temperature, the colloidal solution was filtered through a 0.22 μm nylon filter membrane and stored at 4 °C. Preparation of Amine-Group-Coated Glass Slide Substrates. The preparation of the silanized glass slide substrate was carried out according to the literature.46 The glass slides were first immersed in piranha solution (H2SO4/H2O2 = 3:7) for 1 h and washed thoroughly with ultrapure water and dried under N2 flow. The cleaned substrate was immersed into an ethanolic solution of APTES (1% v/v) for 3 h, washed with ethanol and ultrapure water three times, dried with nitrogen, and baked in an oven at 120 °C for 30 min. Then a monolayer of amino groups on the surface was obtained. Asymmetrical Modification of Satellite GNPs. The aminogroup-functionalized substrate (25 mm × 20 mm) was immersed in the gold colloidal solution for 3 h to adsorb the satellite GNPs through electrostatic attraction. After being rinsed three times with water, the AuNP-adsorbed substrate was placed in a solution of 1 mM SH-PEGCOOH overnight. After being washed with ultrapure water and dried under N2 flow, these PEGylated satellite GNPs were removed from the glass via ultrasonic treatment in 1.0 mL of water for 3 min. The nanoparticles were centrifuged (12000 rpm, 20 min), washed, and resuspended in 1.0 mL of water for the next modification.

influence the calculation of kobs; however, we chose the spots with at least nine stepwise drops for better accuracy as the resolution of color changes was lower in living cells. The cumulative probability of strand displacement is shown in Figure 5c as a function of time. Fitting the curve yielded kobs to be 2.68 × 10−4 s−1. Using the linear fitted rate constants in cell lysis as a reference, the concentration of miRNA-21 in HeLa cells was estimated to be around 2.14 nM. The same batch of HeLa cells was sent for miRNA-21 quantification via RT-PCR with average concentration of 2788 copies per cell. Taking an average volume of the cell as 2.5 pL, we estimated the concentration of miRNA-21 in HeLa to be 1.85 nM, which was close to the result from our experiment. The small gap (14%) might be caused by the different environment of living cells compared to that of the cell lysate; also, it could be experimental error. Anyway, each single core−satellite plasmon ruler could serve as an independent probe to follow the dynamic biological process in living cells. However, compared with the in vitro study, the data acquisition was more difficult because they were always in motion in the living cell, and sometimes we lost the focus on the particles.

CONCLUSION In this work, we demonstrated an optical technique for exploring a DNA/RNA strand displacement process at the single-molecule level in vitro and in vivo. The plasmon rulers were used as probes for continuous observation of toeholdmediated strand displacement, establishing a visualized output in real time with temporal and spatial resolution, which avoids the photobleaching or blinking and provides a platform for continuous monitoring. The one-to-one correspondence of scattering signal to the strand displacement was monitored with great effectivity and precision at the single-molecule level. After information extraction and statistical analysis of a series of DFM images, the kinetics of toehold-mediated strand displacement at the surface of the core GNP were successfully analyzed. The proposed studies revealed the difference in the kinetics of strand displacement between DNA/RNA and DNA/DNA duplexes. The distinct relationship between toehold length and the reaction kinetics for DNA/RNA strand displacement from that of DNA/DNA systems was also observed and studied in detail. We noted that, in different matrixes, there was a big difference with deduced second-order rate constants, indicating the impact of surrounding medium to the displacement kinetics. We also demonstrated the in vivo kinetic study with the core−satellite plasmon rulers in living cancer cells, which helped the determination of the invader (miRNA-21) with spatial resolution. In summary, the core−satellite plasmon rulers along with a dark-field imaging technique offered us a relatively available method for the study of the kinetics in living cells at the single-molecule level. The potential application of this proposed strategy could be further explored as the strand sequence and structure assemblies were alternative and designable. The advantages of unique plasmon rulers enable the monitoring and modulating of dynamical biological processes at the single-molecule level and in living cells, which could not be achieved with a conventional fluorescence method. MATERIALS AND METHODS Reagents. The oligonucleotides used in this work were purchased from Sangon Biological Engineering Technology & Co. Ltd. (Shanghai, China), and RNA invaders were provided by Genepharma. G

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the cells were treated with RIPA lysis buffer III and sonicated at 4 °C. After the complete lysis, the mixture was centrifuged at 12 000 rpm for 5 min at 4 °C, and the supernatant was collected for further use. Cell Culture and Intracellular MiRNA-21 Imaging. Human cervical cancer (HeLa) cells were cultured in DMEM supplemented with 10% FBS and 100 IU·mL−1 penicillin-streptomycin. The cells were maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2). Cells were seeded on a clean glass slide and trapped with a 6 mm holed PDMS as a cell. After 12 h of planting, the culture medium was exchanged with fresh serum-free basal media (500 μL) containing the core−satellite nanoassemblies (30 μL, 50 pM). After 5 h incubation, the cells were washed with 1× PBS three times and then imaged by dark-field microscopy. Finite-Difference Time-Domain Simulation. Three-dimensional full-field FDTD (the package of Lumerical FDTD Solutions 8.15) method was used to perform the simulations about optical properties of core−satellite nanoassemblies. The model was designed as one core GNP and with 12 satellite GNPs surrounding. The distance between core and satellite GNPs is estimated to be 1.2 nm according to the hybridization of complementary DNA strands. The refractive index of the background was set as 1.33, and a total-field scattered-field source, ranging from 400 to 700 nm, was used to investigate the scattering properties of core−satellite assemblies. All of the boundary conditions were all set as perfect, and the mesh size used in the simulating regions was set as small as 0.1 nm. In addition, single-core GNPs and nanoassemblies with different numbers of satellites were also simulated. For simplicity, the contribution of DNA strands and glass slides was ignored. Rate Constant Fitting Procedure. The dynamic toeholdmediated strand displacement reaction has been proven to follow a second-order reaction model:

Preparation of sDNA-Functionalized Satellite GNPs. Thiolated sDNA (0.1 mM) was first activated with 10 mM TCEP in 10 mM Tris-HCl buffer (pH 7.4) for 1 h at room temperature. Then 5 μL of 0.18 μM sDNA was added into 500 μL of asymmetrically PEGylated satellite GNPs. After being incubated under mild shaking for 12 h, the mixture was then aged for another 24 h with NaCl (2.0 M) added to the solution to bring its total NaCl concentration to 0.2 M. Then NPs were centrifuged and washed three times with PBS and resuspended in PBS solution (100 μL, containing 0.1 M NaCl). Then the asymmetrically sDNA-modified satellite GNPs were obtained and stored at 4 °C. Phosphonation and Modification of Core GNPs. The 50 nm core GNPs purchased from Ted Pella, Inc. were first pretreated with BSPP to replace the tannic acid that was capped on the surface of NPs and enhanced the stability of GNPs.47 Briefly, 3 mg of BSPP was added to 10 mL of the colloidal nanoparticle solution under stirring overnight at room temperature. The mixture was centrifuged at 6000 rpm for 15 min, and the GNPs were resuspended in 1 mL of 2.5 mM BSPP solution. After being pretreated with TCEP (10 mM) to reduce the disulfide bonds, the thiol-modified cDNA was incubated with GNPs at the molar ratio of more than 200:1 in PBS for 12 h at room temperature. The mixture was aged for another 24 h, while the concentration of NaCl was gradually increased to 0.2 M. Then the mixture was centrifuged and washed three times with PBS and redispersed in 1 mL of PBS (containing 0.1 M NaCl). Thus, the cDNA-modified core GNPs were obtained. Fabrication of Core−Satellite Nanoassemblies. The cDNAmodified core GNPs and sDNA-modified satellite GNPs were incubated with the molar ratio of 1:25 in PBS (containing 0.1 M NaCl, 5 mM MgCl2) at 37 °C for 3 h. After being centrifuged and rinsed with PBS (10 mM, pH 7.4), the mixture was resuspended in PBS and the core−satellite nanoassemblies were stored at 4 °C for use. Immobilization of Core−Satellite Nanoassemblies on the Substrate. The core−satellite nanoassemblies were diluted and dropped on the surface of silanized glass slides for 3 h and rinsed with PBS. Then the PBS buffer containing sulfo-SMCC (2 mM) was dropped to neutralize the residual surface of extra amino groups for 2 h, followed by washing with PBS. The solution was monitored before and after the addition of the invading strand. DFM Imaging and Scattering Spectra Obtained from Single Core−Satellite Nanoassemblies. The GNP-functionalized slide was immobilized on a platform, and the GNPs were excited by the white light source to generate plasmon resonance scattering light. A true-color digital camera (Olympus DP80, Japan) was used to capture the dark-field color images. The scattering light of the gold nanoparticle was split by a monochromator (Acton SP2358, PI, USA) equipped with a grating (grating density, 300 lines/mm; blazed wavelength, 500 nm) and recorded by an excelon EMCCD (400BR, PI, USA) to obtain the scattering spectra, which were corrected by subtracting the background spectra taken from the adjacent regions without the GNPs and dividing the calibrated response curve of the entire optical system. Fluorescence Experiments for Strand Displacement Kinetics Study. The [CS] double-strand complexes were constructed by prehybridizing the Cy5-labeled cDNA and BHQ2-labeled sDNA in the same molar ratio at 37 °C for 2 h in PBS (pH 7.4, 5 mM Mg2+). After that, 200 μL of diluted [CS] strands and 200 μL of invading strands were mixed together to the final concentration of 10 nM in PBS solution containing 5 mM Mg2+. In this process, the monitoring of fluorescence intensities was carried out upon the addition of the invading strand with time elapsing. In Vitro miRNA-21 Imaging in Cell Disruption Liquid. Human normal bronchial epithelial (BEAS-2B) cells and human cervical cancer (HeLa) cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS and 100 IU·mL−1 penicillin-streptomycin. The cells were maintained at 37 °C in a humidified atmosphere (95% air and 5% CO2). For in vitro experiment with cell lysis solution, BEAS-2B cells were first removed from the culture bottle with trypsin solution, washed with 1× PBS twice, and centrifuged to discard the supernatant. Then

k

CS + I → C + IS

(4)

where k is defined as the displacement rate constant. The reaction could be modeled by the following differential equation:

−

d[CS] d[I] =− = k[CS][I] dt dt

(5)

Compared to the CS double strand, the amount of the invader ([I]) was substantially in excess, and during the whole process, it remained constant, and eq 5 becomes [CS] = [CS]0 exp(− [I]kt )

(6)

which fits into the first-order kinetic equation. We can multiply the reaction rate constant k with assumed constant [I] and define it as an apparent rate constant kobs.

kobs = k[I]

(7)

The cumulative probability (P) of strand displacement was calculated by dividing the number of satellite leaving events up to a given time by the total number of events observed during the experimental time course. On the basis of eqs 6 and 7, the expression of P was established as

P≡

[CS]0 − [CS]t = 1 − exp(− kobst ) [CS]0

(8)

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b08673. Sequences of oligonucleotides used in this work, characterization of the plasmon rulers, plasmonic response of the plasmon rulers, comparison of the kinetics obtained from plasmon rulers and fluorescence method, and kinetics study of strand displacement (toehold length setup, comparison between DNA/ H

DOI: 10.1021/acsnano.7b08673 ACS Nano XXXX, XXX, XXX−XXX

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(15) Zhan, P.; Dutta, P. K.; Wang, P.; Song, G.; Dai, M.; Zhao, S. X.; Wang, Z. G.; Yin, P.; Zhang, W.; Ding, B.; Ke, Y. Reconfigurable Three-Dimensional Gold Nanorod Plasmonic Nanostructures Organized on DNA Origami Tripod. ACS Nano 2017, 11, 1172−1179. (16) Amodio, A.; Adedeji, A. F.; Castronovo, M.; Franco, E.; Ricci, F. pH-Controlled Assembly of DNA Tiles. J. Am. Chem. Soc. 2016, 138, 12735−12738. (17) Gerasimova, Y. V.; Kolpashchikov, D. M. Towards a DNA Nanoprocessor: Reusable Tile-Integrated DNA Circuits. Angew. Chem., Int. Ed. 2016, 55, 10244−10247. (18) Zhang, D. Y.; Hariadi, R. F.; Choi, H. M.; Winfree, E. Integrating DNA Strand-Displacement Circuitry with DNA Tile SelfAssembly. Nat. Commun. 2013, 4, 1965. (19) Machinek, R. R.; Ouldridge, T. E.; Haley, N. E.; Bath, J.; Turberfield, A. J. Programmable Energy Landscapes for Kinetic Control of DNA Strand Displacement. Nat. Commun. 2014, 5, 5324. (20) Zhang, D. Y.; Turberfield, A. J.; Yurke, B.; Winfree, E. Engineering Entropy-Driven Reactions and Networks Catalyzed by DNA. Science 2007, 318, 1121−1125. (21) Yang, L.; Wang, H.; Fang, Y.; Li, Z. Polarization State of Light Scattered from Quantum Plasmonic Dimer Antennas. ACS Nano 2016, 10, 1580−1588. (22) Zhang, L.; Li, Y.; Li, D. W.; Jing, C.; Chen, X.; Lv, M.; Huang, Q.; Long, Y. T.; Willner, I. Single Gold Nanoparticles as Real-Time Optical Probes for the Detection of NADH-Dependent Intracellular Metabolic Enzymatic Pathways. Angew. Chem., Int. Ed. 2011, 50, 6789−92. (23) Shi, L.; Jing, C.; Ma, W.; Li, D. W.; Halls, J. E.; Marken, F.; Long, Y. T. Plasmon Resonance Scattering Spectroscopy at the SingleNanoparticle Level: Real-Time Monitoring of a Click Reaction. Angew. Chem., Int. Ed. 2013, 52, 6011−4. (24) Shi, L.; Jing, C.; Gu, Z.; Long, Y. T. Brightening Gold Nanoparticles: New Sensing Approach based on Plasmon Resonance Energy Transfer. Sci. Rep. 2015, 5, 10142. (25) Lee, S. E.; Chen, Q.; Bhat, R.; Petkiewicz, S.; Smith, J. M.; Ferry, V. E.; Correia, A. L.; Alivisatos, A. P.; Bissell, M. J. Reversible AptamerAu Plasmon Rulers for Secreted Single Molecules. Nano Lett. 2015, 15, 4564−4570. (26) Li, X.-L.; Zhang, Z.-L.; Zhao, W.; Xia, X.-H.; Xu, J.-J.; Chen, H.Y. Oriented Assembly of Invisible Probes: Towards Single mRNA Imaging in Living Cells. Chem. Sci. 2016, 7, 3256−3263. (27) Reinhard, B. M.; Sheikholeslami, S.; Mastroianni, A.; Alivisatos, A. P.; Liphardt, J. Use of Plasmon Coupling to Reveal the Dynamics of DNA Bending and Cleavage by Single EcoRV Restriction Enzymes. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 2667−2672. (28) Qian, R. C.; Cao, Y.; Long, Y. T. Binary System for MicroRNATargeted Imaging in Single Cells and Photothermal Cancer Therapy. Anal. Chem. 2016, 88, 8640−8647. (29) Qian, G. S.; Zhang, T. T.; Zhao, W.; Xu, J. J.; Chen, H. Y. Single-Molecule Imaging of Telomerase Activity via Linear Plasmon Rulers. Chem. Commun. 2017, 53, 4710−4713. (30) Lerch, S.; Reinhard, B. M. Quantum Plasmonics: Optical Monitoring of DNA-Mediated Charge Transfer in Plasmon Rulers. Adv. Mater. 2016, 28, 2030−2036. (31) Jun, Y. W.; Sheikholeslami, S.; Hostetter, D. R.; Tajon, C.; Craik, C. S.; Alivisatos, A. P. Continuous Imaging of Plasmon Rulers in Live Cells Reveals Early-Stage Caspase-3 Activation at the SingleMolecule Level. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 17735− 17740. (32) Volinia, S.; Calin, G. A.; Liu, C. G.; Ambs, S.; Cimmino, A.; Petrocca, F.; Visone, R.; Iorio, M.; Roldo, C.; Ferracin, M.; Prueitt, R. L.; Yanaihara, N.; Lanza, G.; Scarpa, A.; Vecchione, A.; Negrini, M.; Harris, C. C.; Croce, C. M. A MicroRNA Expression Signature of Human Solid Tumors Defines Cancer Gene Targets. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 2257−2261. (33) Iorio, M. V.; Ferracin, M.; Liu, C. G.; Veronese, A.; Spizzo, R.; Sabbioni, S.; Magri, E.; Pedriali, M.; Fabbri, M.; Campiglio, M.; Menard, S.; Palazzo, J. P.; Rosenberg, A.; Musiani, P.; Volinia, S.; Nenci, I.; Calin, G. A.; Querzoli, P.; Negrini, M.; Croce, C. M.

DNA and DNA/RNA, influence from surrounding medium) (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jing-Juan Xu: 0000-0001-9579-9318 Author Contributions †

M.-X.L. and C.-H.X. contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully acknowledge the National Natural Science Foundation (Grants 21327902, 21535003, and 21605079) of China, and the Natural Science Foundation (BK20160637) of Jiangsu Province. This work was also supported by National Key R&D Program of China (Grant No. 2016YFA0201200), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. REFERENCES (1) Zhang, D. Y.; Seelig, G. Dynamic DNA Nanotechnology Using Strand-Displacement Reactions. Nat. Chem. 2011, 3, 103−13. (2) Zhang, D. Y.; Winfree, E. Control of DNA Strand Displacement Kinetics using Toehold Exchange. J. Am. Chem. Soc. 2009, 131, 17303−14. (3) Qian, L.; Winfree, E. Scaling up Digital Circuit Computation with DNA Strand Displacement Cascades. Science 2011, 332, 1196−1201. (4) Penchovsky, R.; Breaker, R. R. Computational Design and Experimental Validation of Oligonucleotide-Sensing Allosteric Ribozymes. Nat. Biotechnol. 2005, 23, 1424−33. (5) Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. EnzymeFree Nucleic Acid Logic Circuits. Science 2006, 314, 1585−1588. (6) Soloveichik, D.; Seelig, G.; Winfree, E. DNA as a Universal Substrate for Chemical Kinetics. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 5393−8. (7) Zhang, D. Y.; Winfree, E. Dynamic Allosteric Control of Noncovalent DNA Catalysis Reactions. J. Am. Chem. Soc. 2008, 130, 13921−13926. (8) Zhang, D. Y.; Turberfield, A. J.; Yurke, B.; Winfree, E. Engineering Entropy-Driven Reactions and Networks Catalyzed by DNA. Science 2007, 318, 1121−1125. (9) Zhang, D. Y.; Winfree, E. Robustness and Modularity Properties of a Non-Covalent DNA Catalytic Reaction. Nucleic Acids Res. 2010, 38, 4182−97. (10) You, M.; Chen, Y.; Zhang, X.; Liu, H.; Wang, R.; Wang, K.; Williams, K. R.; Tan, W. An Autonomous and Controllable LightDriven DNA Walking Device. Angew. Chem., Int. Ed. 2012, 51, 2457− 60. (11) Muscat, R. A.; Bath, J.; Turberfield, A. J. A Programmable Molecular Robot. Nano Lett. 2011, 11, 982−7. (12) Tomov, T. E.; Tsukanov, R.; Glick, Y.; Berger, Y.; Liber, M.; Avrahami, D.; Gerber, D.; Nir, E. DNA Bipedal Motor Achieves a Large Number of Steps due to Operation using Microfluidics-based Interface. ACS Nano 2017, 11, 4002−4008. (13) Green, S. J.; Bath, J.; Turberfield, A. J. Coordinated Chemomechanical Cycles: a Mechanism for Autonomous Molecular Motion. Phys. Rev. Lett. 2008, 101, 238101. (14) Wickham, S. F.; 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. I

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DOI: 10.1021/acsnano.7b08673 ACS Nano XXXX, XXX, XXX−XXX