An Exploration of the Kinetics of Toehold Mediated Strand

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An Exploration of the Kinetics of Toehold Mediated 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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08673 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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An Exploration of the Kinetics of Toehold Mediated 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. † These authors contributed equally to the work. * To whom correspondence should be addressed. E-mail: [email protected] (J.-J.X); [email protected]. (W.Z.)

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ABSTRACT: DNA/RNA strand displacement is one of the most fundamental reaction 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 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 was proceeded for the first time, which was not possible by conventional method with fluorescent reporter. The plasmon rulers, which are flexible, easily constructed and robust, have proven to be effective tool in exploring the dynamical behaviors of biochemical reactions in vivo.

KEYWORDS: plasmon rulers, dark-field microscopy, strand displacement, kinetics, singlemolecule imaging,

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 reaction in dynamic DNA nano-devices, is usually initiated by a short sequence of single strand domain names as toehold and causes the displacement of one strand of


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DNA of another in binding to a third strand with partial complementarity to both.2 Such enzymefree 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 non-covalent DNA catalysis,7-9 autonomous DNA nanomachines10-14 as well as reconfiguration of DNA nanostructures15-18 at the nanoscale. The kinetics of toehold mediated strand displacement is 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 toehold, and it was possible to predict the kinetics of different strand displacement reactions and impact of mismatches on 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 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 continuously imaging for hours.26 Via recording the wavelength shifts or intensity changes of the plasmonic probes, some important biological process such as DNA bending and cleavage,27 RNA digestion,28 activity of single telomerase,29 and single-DNA hybridization30 have been read out. Since the kinetics of


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stand displacement strongly depends on the matrix, to build models both in vitro and in living cells, ‘plasmon rulers’ could be suitable candidates.

Scheme 1. Schematic illustration of experimental design and process of toehold mediated strand displacement. Herein, inspired by the continuous imaging of plasmon rulers to measure dynamic biophysical processes,31 we synthesized a core-satellite gold nanoparticles assembly and investigated the kinetics of toehold mediated strand displacement at the surface of gold nanoparticle (GNP). 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 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 additional reaction, the kinetics of strand displacement could be studied at single molecule level. We chose microRNA-21 (miRNA-21), the oncogene as the invading strand since 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


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DNA/DNA duplexes, and help 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 opportunity in kinetics study in vivo, or under extremely low concentration (at single molecule level), which couldn’t 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 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) bond to the core via DNA strand hybridization. As shown in Scheme 1b, the core GNP was functionalized with complementary DNA (cDNA) that comprised of α and β domain, in which the β domain was complementary to the β 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 most possibly bonded to the core through single DNA duplex. Detailed determination process is shown in the Supporting Information. According to dynamic light scattering (DLS) measurements, hydrodynamic diameters of satellite GNP, core GNP and core-satellite nanostructure were around 16.5 nm, 51.5 nm and 85.7 nm, respectively (Figure S1d). The structure of core-satellite nanoassembly was further characterized using high-resolution transmission electron microscope (HR-TEM) (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 satellites number ranges from 8-12, mainly at 10 and 11 (75%).


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However, it was hard to precisely estimate the distance between core and satellite GNPs from these TEM images, since the 3D core-satellite structure was rearranged to 2D-packed structure during drying process.40 To make accurate estimation, we applied GNPs with equal size (50 nm in diameter) and synthesized GNP-dimer using the same ssDNA sequences (cDNA and sDNA) as for the core-satellite nanoassembly. Since two GNPs of the dimer are lying in the same plane on 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 spectrometer. As shown in Figure 1, core GNPs modified with cDNA were observed as dark green spots with scattering wavelength centered at 560 nm. The modification of DNA strands changed the dielectric layer around the nanoparticle,41 resulting in slight red-shift and change in the peak shape (Figure S7). While the core-satellite assemblies scattered bright salmon pink spots with λmax of 622 nm. Compared with core GNP, the core-satellite nanoassembly exhibited highly increased scattering intensity (ca. 10 times), and a significant red shift (∆λ= 62 nm), which made such probe excellent plasmon ruler for the study of dynamic process. From theoretical simulation using the finite-difference time-domain (FDTD), ∆λ of 68 nm was estimated in comparison of core GNP (λmax= 544 nm) with 12 satellites assembly (λmax= 612 nm) (Figure S8), which 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 total-field scattered-field source, two scattering peaks are shown for the GNP core-satellite nanoassembly. However, the


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obtained spectrum is composed of a sum of the two peaks, which couldn’t be simply fitted with one Lorentzian line shape.

Figure 1. Core-satellite nanoparticle plasmon rulers. The scattering image of separated cDNA modified core GNPs (a) and core-satellite nanoassemblies (b) under DFM, and the scale bar of inset TEM image is 20 nm. The scattering spectra (c) of a single nanoassembly (red) and a single core GNP (black). Kinetic mode of strand displacement process. The toehold-mediated strand displacement at the surface of core GNP was described by the three-step model2 (Scheme 1b) .The hybridized double strand is composed of sDNA (S) with toehold domain (γ) and complementary DNA (C). The invading strand abbreviated as stand I consisted of β and γ domains, in which γ domain was the toehold complementary sequence (Table 1). Firstly, invading strand I bond to the exposed toehold γ , then after the branch migration, β domain of complementary DNA was displaced by the invader with same oligonucleotides sequence. Finally, complementary DNA was released, while a new double strand of I+S was formed. Each strand displacement event made one satellite leaving from the core, and could induce shifting of scattering wavelength (Figure S8). In other words, the strand displacement process could be monitored via the scattering spectra of plasmon


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rulers at single molecule level for kinetics study. As previously reported, in sufficient low concentration, the kinetics of toehold-mediated strand displacement are well-approximated 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 → C + IS CS + I 

(1)

where k is the second-order rate constant. In this system, compared to CS double strand (0.4 pM in 30 µL reservoir), the amount of the invader (0.1 nM - 200 nM) was substantially excess, which kept constant during the whole displacement process. The process appears as a pseudofirst-order reaction. We defined apparent rate constant kobs by multiplying the displacement rate constant k with constant concentration [I].

kobs = k [ I]

(2)

Detailed rate constant fitting procedure is demonstrated in method. In conventional fluorescent measurements, an additional reporter system is needed, in which the displaced strand displaces a fluorescently labelled 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:

P = 1 − exp ( − kobst )

(3)


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The apparent rate constant kobs could be deduced based on the observed P. Also, the second-order constant k could be fitted, with reference value compared to the results from conventional florescence method. Table 1. Domain sequences domain

sequences

length (nt)

α

5’ -TTT -3’

β

5’ - TCAGACTGATGTTGA-3’

γ

5’ -TAGCTTA -3’

3 15 7

Analyses of plasmon rulers for strand displacement. We first investigated the dynamical strand displacement process under in vitro conditions of 10 mM PBS buffer under room temperature. The core-satellite nanoassemblies were immobilized on the surface of an amino group functionalized silane-treated glass, covered with ultra-thin 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 adding 5.0 nM invader (miRNA-21), initial bright salmon pink spots turned to yellow and then dark green as time elapsed (Figure 2b). The scattering spectra of the selected signal-assembled nanoparticle was also monitored exactly after every DFM image was taken (Figure 2c). The scattering wavelength showed a stepwise blueshifts in corresponding to individual strand displacement event (Figure 2d). Every strand displacement event would cause the wavelength blue-shifted for 5 nm on average. As the scattering intensity and wavelength changed synchronously with the R/G drops, but color analysis was much easier, we chose the stepwise decrease of R/G to indicate the strand displacement events for kinetics study.


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Figure 2. DFM imaging and spectra analyses of plasmon rulers. (a) Strand displacement mediated plasmon rulers. (b) A series of DFM images monitored after the addition of invading strand (scale bar: 5 µm). (c) The scattering spectra of the selected nanoassembly in the strand displacement. (d) The intensity changes (∆I = I1 - I0, where I0 means the original intensity value and I1 means the intensity at time t1 during the displacement) and wavelength shifts (∆λ = λ1-λ0, where λ0 means the original peak wavelength and λ1 means the peak wavelength at time t1 during the displacement). 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 red/green (R/G) value with certain waiting time (tw) were observed at one single spot. Meanwhile, the gray value also


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decreased step by step with time elapsing (Figure S9), indicating the dimming of scattering intensity. In control experiments with addition of 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 the stepwise signal changes were from satellite GNPs’ leaving events. In an ideal mode, each step indicated a single strand displacement event. But in the experiment, 2 or more strands might displace in the same detecting time, resulted in a larger height of steps (∆ R/G). In statistical study, we took 4 concentrations (0.5 nM, 1.0 nM, 5.0 nM and 50.0 nM) of the invader and 40 plasmon rulers in total to evaluated ∆ R/G of 380 steps. According to the distribution, 0.045 was defined as the ∆ R/G caused by a single event (Figure 3b). The exact number of events up to a given time was calculated by dividing the recorded ∆ R/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 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 rulers 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 (∆R/G) were assessed during different periods of time. As shown in Figure S11, with 10 nM invader, the averaged ∆R/G in three time periods are 0.049, 0.041 and 0.045, respectively. Therefore, the scattering spectra response was approximately equal with different number of satellites in the experiments.


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Figure 3. R/G value analyses for strand displacement events. (a) R/G value trace (black dots) and fitted stepwise signal (red line) of single nanoassembly with time elapsing (inset: R/G value trace of displacement process with miRNA-141 as invader strand). (b) The distribution of R/G value changes for every step height during the strand displacement process. (c) Statistical analyses of satellite GNPs’ leaving events from the core with different concentration (0.1-200 nM) of invader as a function of time. In vitro studies of strand displacement kinetics. Based on the statistical events distribution of 20 plasmon rulers and more than 200 events (Figure 3c), the P value of strand displacements (7nt toehold length) was calculated and fitted with equation 3 (Figure 4a). The fitting curves yielded kobs (0.61×10-4s-1 - 3.98×10-4s-1) at different concentrations of the invader. In sufficient 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-1s-1. However, when the concentration of miRNA-21 was larger than 2.0 nM, the linear correlation between kobs and the concentration became worse. Since the concentrations of intermediated products haven’t been accounted, the concentrations of


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[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 that obtained using conventional fluorescent method. Since the length of 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 florescence photometer. At first, we tried to use low concentrations (< 1nM) of CS double strand to simulate the condition as close as possible. However, the signal to noise ratio was too large to get convinced experimental results. Therefore, finally the concentrations of both [CS] complex and invader strands were set as 10 nM, and the second-order constant k was fitted as 7.14×105 M-1s-1. Compared with k deduced from plasmon ruler (1.31×105 M-1s-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 BM model. Since 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 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 oppotunity in


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kinetics study under extremely low concentration (at single molecule level), which couldn’t be achieved using conventional fluorescence method. The kinetics study with three different lengths of toehold (6nt, 7nt and 9nt) was also performed. As shown in Figure S13, the fitted reaction constants are 8.62×104 M-1s-1, and 1.38×105 M-1s-1 with 6nt and 9nt, respectively. From the results, it was found reaction rate of the toehold-mediated strand displacement reaction saturated for a toehold of 7nt. 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 6nt2 and 4nt43, respectively. As well known, for short toeholds, the invading strand would fall off from the toehold and undergo frequent binding and unbinding from the toehold. For long toeholds, the probability of falling off becomes smaller, hence it will successfully complete displacement with a high probability. As previous studies reported,44 the thermodynamic stability of duplexes is in order of RNA/RNA > DNA/DNA > hybrids, it is reasonable to conclude that the strand displacement of DNA/RNA needs longer toehold length since 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 shown in Figure S14, which confirmed the lower thermodynamic stability of duplex than DNA/DNA. We also examined DNA/DNA strand displacement with same sequences as DNA/RNA, the displacement rate constant k was fitted to be 3.26×105 M-1s-1, which was much higher than that of DNA/RNA (1.31×105 M-1s-1). Fluorescence experiments confirmed the correctness of the conclusion (Figure S13). We tried to explain such phenomenon from both the secondary structures of DNA and RNA invader strands, and the thermodynamic stabilities of formed DNA/DNA and DNA/RNA duplexes. Since the strand displacement requires opening of the stem


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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 DNA strand with the same sequences, which indicated that more energy would be needed to break the relatively stable secondary structure of microRNA-21 in the solution.44 Further, as previously mentioned, the stability of hybrids is lower than DNA duplex, therefore, it is reasonable to expect a higher reaction rate constant for the formation of DNA/DNA duplexes than that for hybrids.

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.


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Intracellular studies of strand displacement kinetics. The typical fluorescence kinetics experiment processed in relative simple system. However, the surrounding medium could greatly influence the dynamic process of strand displacement. Before kinetics study in vivo, we took cell lysate of BEAS-2B cells, human normal bronchial epithelial cells, to simulate physiological environment of living cells, and miRNA-21 was put in the medium by standard addition. kobs of strand displacement based on 20 plasmon rulers was fitted. As Figure S16 depicted, kobs was liner to the concentration of miRNA-21 with the range from 0.1 nM to 2.5 nM, and k was fitted as 9.68×104 M-1s-1 (R=0.997), which was 25% lower than that from 10 mM PBS buffer. The result suggests that in physiological environment like living cells, to make accurate prediction of a dynamic process, calibration factor needs to be considered. The nanoassemblies with unlimited lifetime were introduced into human cervical cancer (Hela) cells for the real-time detection with single particle resolution. For in situ imaging of intracellular miRNA-21, Hela cells were seeded in a clean glass slide for 24 h, followed by the delivery of core-satellite nanoassemblies. After incubation of the plasmon rulers for several hours, salmon pink spots appeared in the cytoplasm of the cells under DFM, then the solution of plasmon rulers was removed and replaced by 10 mM PBS. The outlines of cells were profiled with dashed lines, and two spots selected to be monitored were amplified (Figure 5a). Similar stepwise drops of R/G were observed compared with in-vitro observation, and we made the distribution of strand displacement events derived from 13 probes as a function of time (Figure 5b). According to the fitting equation 3, the chosen of initial observation time won’t influence the calculation of kobs, however, we chose the spots with at least 9 stepwise drops for better accuracy since 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


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2.68×10-4s-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. Same batch of Hela cells were sent for miRNA-21 quantification via real time PCR with average concentration of 2788 copies per cell. Taking an average volume of the cell as 2.5 pL, the concentration of miRNA-21 in Hela was estimated 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 cell lysate, also, it could be experiment error. Anyway, each single core-satellite plasmon ruler could serve as an independent probe to follow dynamic biological process in living cells. However, compared with in-vitro study, the data acquisition was harder since they were always in motion in the living cell, sometimes we lost the focusing on the particles.


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Figure 5. Imaging and analyses of plasmon ruler in living cells. (a) DFM images monitored after the introduction of core-satellite nanoassemblies in living Hela cells with time elapsing (scale bar: 10 µm). (b) Statistical analyses of satellite GNPs’ leaving events from the core in cells as a function of time. (c) The cumulative probability of strand displacement as a function of time.

CONCLUSION In this work, we demonstrated an optical technique in exploring DNA/RNA strand displacement process at single-molecular level in vitro and in vivo. The plasmon rulers were used as probes for continuous observation of toehold mediated strand displacement, establishing an optical visualized output in real-time with temporal and spatial resolution, which avoid the


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photobleaching or blinking and provided a platform for continuously monitoring. The one-to-one correspondence of scattering signal to the strand displacement were monitored with great effectivity and precision at single molecular level. After information extraction and statistical analysis of a series of DFM images, the kinetics of toehold mediated strand displacement at the surface of core GNP was 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 of 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. To summary, the core-satellite plasmon rulers along with dark field imaging technique offered us a relatively available method for the kinetics study in living cells at single molecular level. The potential application of this proposed strategy could be further explored since 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 single molecule level, and in living cells, which couldn’t be achieved with 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 was provided by Genepharma. All the sequences are shown in Table S1. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·4H2O) and sodium citrate were obtained from Sinopharm Chemical Reagent


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Co.,

Ltd.

Tris

(2-carboxyethyl)

aminopropyl)triethoxysilane

(APTES,

phosphine 99%),

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hydrochloride

(TCEP),

(3-

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-N-hydroxysuccinimide 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 pretreated with diethylpyrocarbonate and used throughout the experiment. 1×PBS buffer used in the cell culture was purchased from Key GEN Biotech, and other buffer solutions used in the experiments were prepared as follows: PBS buffer (10 mM Na2HPO4/NaH2PO4, pH=7.4), Tris-HCl buffer (10 mM Tris, pH=7.4). Apparatus. Transmission electron microscopy (TEM) was performed with JEM-1011(JEOL Ltd., Japan). High-resolution electron microscopy (TEM) 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 (DLS) 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


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(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 (GNPs). The satellite gold nanoparticles with 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 (3 parts concentrated HCl, 1 part concentrated HNO3), rinsed with ultrapure water, and oven-dried prior to use. 50 mL 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 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 slides substrate. The preparation of silanized glass slides 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 respectively for 3 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 amino group 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 3 times with water, the AuNP-adsorbed substrate was placed in a solution of 1 mM SH-PEG-COOH overnight. Washed with ultrapure


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water and dried under N2 flow, these PEGylated satellite GNPs were removed from the glass via ultrasonic treatment in 1.0 mL water for 3 min. The nanoparticles were centrifuged (12000 rpm, 20 min) and washed, then resuspended in 1.0 mL water for the next modification. Preparation of sDNA functionalized satellite GNPs. 0.1 mM thiolate sDNA 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 asymmetrically PEGylated satellite GNPs. After incubated under mild shaking for 12 h, the mixture was then aging 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 buffer, and resuspended in PBS buffer solution (100 µL, containing 0.1 M NaCl). Then the asymmetrically sDNA modified satelliteGNPs 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 capped on the surface of NPs and enhance the stability of GNPs.47 Briefly, 3 mg BSPP was added to 10 mL of the colloidal nanoparticles 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 more than 200:1 in PB buffer for 12 h at room temperature. The mixture was aging for another 24 h while the concentration of NaCl was gradually increased to 0.2 M. Then the mixture was centrifuged and washed for 3 times with PBS buffer, and redispersed in 1 mL PBS buffer (containing 0.1 M NaCl). Thus, the cDNA modified core-GNPs were obtained.


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Fabrication of core-satellite nanoassemblies. The cDNA modified core-GNPs and sDNA modified satellite-GNPs were incubated with the molar ratio of 1:25 in PBS buffer (containing 0.1 M NaCl, 5 mM MgCl2) at 37 °C for 3 h. After being centrifugated and rinsed with PBS buffer (10 mM, pH 7.4), the mixture was resuspended in PBS buffer and the core-satellite nanoassemblies were stored at 4 °C for use. Immobilization of core-satellite nanoassemblies on substrate. The core-satellite nanoassemblies were diluted and dropped on the surface of silanized glass slides substrate for 3 h and rinsed with PBS buffer. 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 buffer. The monitor was carried out before and after the addition of invading strand. DFM imaging and scattering spectra obtaining of single core-satellite nanoassemblies. The GNP-functionalized slide was immobilized on a platform, and the GNPs was 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 was corrected by subtracting the background spectra taken from the adjacent regions without the GNPs and dividing with the calibrated response curve of the entire optical system. Fluorescence experiments for strand displacement kinetics study. The [CS] double-strands complex were constructed by pre-hybridizing 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


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Page 24 of 34

concentration of 10 nM respectively in PBS solution containing 5 mM Mg2+. In this process, the monitoring of fluorescence intensities was carried out upon the addition of 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 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 buffer for twice and centrifuged to discard the supernatant. Then the cells were treated with RIPA lysis buffer III and sonication at 4 °C. After the completely lysis, the mixture was centrifuged at 12000 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 medium supplemented with 10% FBS and 100 IU·mL-1 penicillinstreptomycin. 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 (FDTD) simulation. Three-dimensional full-field finitedifference time domain (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 twelve satellite GNPs around. The distance between


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core and satellite GNPs is estimated as 1.2 nm according to the hybridization of complementary DNA strands. The refractive index of 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 coresatellite assemblies. All of the boundary conditions were all set as perfect and the meshing size used in the simulating regions was set as small as 0.1 nm. Besides, single core GNP and nanoassemblies with different number of satellites were also simulated, respectively. For simplicity, the contribution of DNA strands and glass slide were ignored. Rate constant fitting procedure. The dynamic toehold-mediated strand displacement reaction has been proven to follow a second-order reaction model: k → C + IS CS + I 

(4)

k was defined as the displacement rate constant. The reaction could be modelled by the following differential equation: -

d [ CS] d [I] == k [ CS][ I ] dt dt

(5)

Compared to CS double strand, the amount of the invader ([I]) was substantially excess, and during the whole process it kept constant, and equation 5 becomes:

[CS] = [CS]0 exp(−[I]kt )

(6)

which fits to the first-order kinetic equation. We can multiply the reaction rate constant k with assumed constant [I], and defined it as 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


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Page 26 of 34

experimental time-course. On the basis of equation 6 and 7, the expression of P was established as: P≡

[ CS] − [CS] [CS] 0

t

= 1 − exp ( −kobst )

(8)

0

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. 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/DNA and DNA/RNA, influence from surrounding medium).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. (J.-J.X); [email protected]. (W.Z.) Author Contributions †M.-X.L.

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

ORCID Jing-Juan Xu: 0000-0001-9579-9318


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ACKNOWLEDGMENT We gratefully acknowledge the National Natural Science Foundation (Grants 21327902, 21535003, 21605079) of China, Natural Science Foundation (BK20160637) of Jiangsu Province. This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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For TOC only:

An exploration of the kinetics of the toehold mediated strand displacement process via coresatellite plasmon rulers in vitro and in living cells at single-molecule level.


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An Exploration of the Kinetics of Toehold Mediated Strand

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