Stochastic RNA Walkers for Intracellular MicroRNA Imaging

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Stochastic RNA Walkers for Intracellular MicroRNA Imaging Mingshu Xiao, Xiwei Wang, Li Li, and Hao Pei Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02265 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 12, 2019

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Analytical Chemistry

Stochastic RNA Walkers for Intracellular MicroRNA Imaging Mingshu Xiao, Xiwei Wang, Li Li, and Hao Pei* Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, P. R. China ABSTRACT: Nucleic acid-based machines have sparked tremendous attention because of their potential applications in biosensing, drug delivery, and biocomputing. Herein, we construct an enzyme-propelled stochastic RNA walker that autonomously walks on single wall carbon nanotube (SWCNT)-based one-dimensional (1D) track for miRNA imaging in living cells. Driven by duplex-specific nuclease (DSN) with capability of selective digestion of DNA in the DNA/RNA heteroduplexes, the RNA walker enables autonomous and progressive walk on the SWCNTs, producing amplified signal outputs. As a result, this DSN-powered stochastic RNA walker with high target-recycling kinetic achieves prominent detection performance of miRNA analysis, showing a linear range from 5 fM to 10 pM with a limit of detection of 1.67 fM and one-base mismatch discrimination. Finally, we demonstrated that this nanomachine can be applied for intracellular miRNA imaging.

Molecular machines can carry out various sorts of nontrivially autonomous functions (e.g., intracellular transport, molecular computing, signal transduction) in response to specific external stimuli.1-3 As a highly programmable means, DNA self-assembly has elicited much interest in constructing such intelligent man-made systems (e.g., tweezers, motors, robots, molecular transporter) because of the exquisite specificity, predictability, and diversity of DNA hybridization.4-7 Among them, tremendous attention has been attracted on developing intelligent, processive nucleic acid-based walking machines in last decade,8, 9 whose mechanical motions are powered by chemical energy in principle.10 Accumulating evidence has demonstrated that these engineered walking machines propelled by enzymes or strand displacement can move along precisely designed one-, two- and three-dimensional (1, 2, and 3D) tracks.11, 12 More importantly, these walkers have been reported to transduce and quantify signals from isothermal molecular amplifications.13, 14 For instance, a single target-binding event induces release of hundreds of signal probes from an enzyme-propelled DNA walker, thereby leading to amplified signal outputs and enhancing detection sensitivity.15 Therefore, nucleic acid-based walking machines have been applied as novel biosensors for sensitive detection.16-18 MicroRNAs (miRNAs) as post-transcriptional regulators can suppress the expression of target messenger RNAs and thus reduce the protein level,19, 20 which makes them play an important role in diverse biological processes, such as cell proliferation, differentiation, apoptosis, and metabolic homeostasis.21-23 In addition, their dysregulated expressions are often related to cancer development and progression and resistance to therapy because miRNAs function either as oncogenes or as tumor suppressors.24, 25 MiRNA is thus considered as a class of valuable biomarkers for cancer diagnosis, prognosis and therapy, and drug targets.26-28 As such, the boosted demand provides powerful impetus for scientiﬁc researchers to establish an accurate

and sensitive analytical methodology for monitoring intracellular miRNA levels, which facilitates to understand their biological functions, evaluate drug efficacy, and identify cancerous cells. Despite the fact that numerous methods have been developed for miRNA detection, yet most of them are compromised by the reduced targettriggered hybridization efficiency between sensing probes and target.29-35 Such slow target-recycling kinetics seriously hampers potential bioanalysis of low-abundance miRNAs in living cells. To address the aforementioned challenge, we construct a single wall carbon nanotube (SWCNT)-based nucleic acid walker. The nanomachine powered by enzyme performs automatic and progressive walk and leads to concurrent

Figure 1. Schematic for DSN-propelled RNA walkers moving on one SWCNT.

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mm probe sonicator (Sonics, VCX-500). The suspension was then centrifuged for 1.5 h at 17,000 g to remove impurities and aggregates. Thereafter, the un-adsorbed DNA oligonucleotides were removed via spin filtration for three times. Finally, DNA-wrapped SWCNTs were collected and re-dispersed for further experiment. miRNAs Detection. The sensitive detection of miRNA was carried out in a 100 µL reaction mixture containing 0.1 U DSN, 20 U RNase inhibitor, 1× DSN master buffer (50 mM Tris-HCl (pH 8.0), 5 mM MgCl2 and 1 mM DTT), and different concentrations of target miRNA. Following, the prepared SWCNT-based probes were added into the reaction with final concentration of 10 g/mL. The mixture was incubated at 40 °C for 30 min before fluorescence detection. TIRF Imaging and Analysis. The cover slides were successively cleaned with NaOH (2M), hellmanex (2%), and ethanol (99%), respectively. Then, the cleaned slides were incubated in a solution of amino-PEG-silane, followed by washing with ddH2O and drying with N2. The SWCNT-based probes were deposited on the cleaned slide for 10 min, and washed with 1PBS buffer. For the walking device, a solution of RNA walking strand (10 nM) and SWCNT-based probes (10 g/mL) was mixed and incubated for 15 min at 37 oC. Fluorescent images were captured with a commercial TIRF microscopy (Nikon), in which a 100× oilimmersion objective and 488- nm laser was used. After addition of DSN, the fluorescence intensity changing over time was recorded. For single particle tracking, each frame of the fluorescence series image was analyzed using ImageJ software (US National Institutes of Health) with a manual tracking plugin. Cell Culture. HeLa cells were cultured in 25 cm2 cell culture flask containing Dulbecco's Modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin at 37 ºC in a humidified 5% CO2 incubator. Preparation of the Reaction Mixture for the Transfection of SWCNT-Based Probe and DSN. In typical, preparation of the reaction mixture was as follows: first, the A solution (120 L Opti-MEM, 12 L probe (5 g/mL), and 0.4 U DSN) and the B solution (120 L Opti-MEM and 12 L liposome-2000 (1 mg/mL)) were incubated separately for 10 min at room temperature. Then, A solution and B solution were mixed and incubated for 20 min at room temperature, yielding A/B mixture. Investigation of Cytotoxicity of SWCNT-Based Probe, DSN, and Liposomes. To investigate the cytotoxicity of SWCNT-based probes, DSN, and liposomes, 100 L DMEM with 10% FBS containing HeLa cells was seeded into a 96well cell culture plate (1104 cells per well) and incubated for 24 h. 10 L aliquots of the A/B mixture were added into the above 96-well. Thereafter, a 10 L aliquot of CCK-8 reagent was added to one of the above wells at different time points (0, 1, 2, 3, 4, 5, 12, and 24 h), and incubation was continued for another 4 h at 37 °C in a humidified 5% CO2 incubator. After incubation, OD450 of each well was measured by a microplate reader. For the control group, a 10 L aliquot of Opti-MEM without our nanodevice was added to the 96-well plate containing HeLa cells, followed by addition of 10 L of CCK-8 reagent. The wells were incubated for 4 h at 37 ºC in a humidified 5% CO2 incubator and the OD450 was determined.

signal release, allowing for intracellular miRNA imaging. As illustrated in Figure 1, our RNA walker contains three components: 1D SWCNT track, duplex-specific nuclease (DSN), and walking strand (RNA). The 1D track is comprised of 300 to 400-nm-length SWCNT wrapped with DNA diblock oligomers (labeled with gray and green in Figure 1). Single-foot RNA walker is composed of a 22-base sequence complementary to the SWCNT. Upon consumption of DNA-wrapped SWCNT by DSN, the walking strand achieves autonomous walk. Briefly, DSN recognizes the formed DNA/RNA heteroduplexes and selectively digests DNA in heteroduplexes.36, 37 As a result, the released RNA walker subsequently binds to another stand of SWCNT and initiates a next cleavage cycle. Hence, the movement of the RNA walker on such a 1D SWCNT track offers an effective approach to transduce and amplify biorecognition signals. In view of one single-target binding event inducing release of abundant signal probes, this DSN-propelled RNA walker with high target-triggered hybridization efficiency is envisioned to enable sensitive detection of miRNA.

EXPERIMENTAL SECTION Reagents and Materials. Oligonucleotides listed in Table S1 were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China) with standard desalting and purified with high-performance liquid chromatography. DSN was obtained from Newborn Co. Ltd. (Shenzhen, China). CoMoCAT SWCNT was acquired from Southwest Nanotechnologies. Lipofectamine-2000 was purchased from Invitrogen (Grand Island, NY, USA). Glass Bottom Dish (D35-20-1-N) was provided by Xinyou Biotechnology Co., Ltd. (Cellvis, Hangzhou, China). HeLa cells (a human cervical cancer cell line) were provided by the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM, fetal bovine serum (FBS), and trypsin/EDTA (0.25%) were purchased from Gibco (Gaithersburg, USA). CCK-8 reagent was obtained from Dojindo (Japan). All other reagents used in this work were of analytical grade and directly used without further purification. Ultrapure water (18.2 MΩ) purified with a Milli-Q Integral water purification system (Millipore Corp., Bedford, MA) was used throughout the study. Instrumentation. The fluorescence emission spectra were recorded with a HITACHI F-7000 fluorophotometer (Hitachi, Japan) at ambient temperature. The excitation wavelength was set at 485 nm with a 3 nm bandwidth and 0.3 s integration time, and the emission spectra were collected from 500 to 650 nm with a wavelength step of 2 nm/s. The AFM imaging was conducted using the automode on a silicon wafer under air condition with a multimode-8 controller from Bruker Instruments. The TEM image was taken by a JEM-2100F (JEOL, FEI, Japan). Cells were cultured in a cell incubator (Eppendorf Galaxy 170S, Germany). The cells were visualized by a Nikon confocal scanning system. Preparation of DNA-Wrapped SWCNTs. In a typical experiment, 1 mg of CoMoCAT SWCNT powder was dispersed in 1 mL 0.1 M NaCl (containing 1.2 mg/mL A1 DNA oligonucleotide). The sample was sonicated in an icewater bath for 2 h at a nominal power of 3 W/mL using a 3 B


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Analytical Chemistry miRNA Imaging in HeLa Cells. The procedures for miRNA imaging using a Leica confocal scanning system were as follows. 500 L aliquots of DMEM with 10% FBS containing HeLa cells were seeded into a 24-well plate (1105 cells per well) with bottom glass. After incubation for 24 h, the cells were washed twice with 1×PBS. Then, 50 L aliquots of the A/B mixture were added into each well and incubated at 37 °C in a humidified incubator for another 5 h. Cells without treatment were used as control group and cells incubated with SWCNT-based probes and Lipofectamine in the absence of DSN as negative controls. After washing twice with 1PBS, the fluorescence imaging of the cells was carried out with a Nikon confocal scanning system. Flow Cytometry Experiment. The transfected cells were harvested by trypsinization, washed three times with 1×PBS, and suspended in 1PBS to conduct flow cytometry experiments. The cells were processed for imaging flow cytometry and analyzed by an ImageStream mkII system (Merk Serono Co., Ltd, Darmstadt, Germany).

reaching a plateau in 720 s (Figure 2c). On the contrary, SWCNTs showed a little fluorescence loss (15%) in 900 s in the absence of RNA walking strands (Figure 2d). Taking these results together, it is concluded that DSN-powered RNA walker allows for autonomous walk on the SWCNT, producing amplified fluorescence signals.

RESULTS AND DISCUSSION Construction of the SWCNT-Based 1D Track. The SWCNT-based 1D track was built by wrapping SWCNTs with single-strand DNA (ssDNA).38 The ssDNA contains two domains—one with (GT)10 for encapsulating SWCNTs (SWCNT-binding sequence)39 and a second with a sequence complementary to a target miRNA (miRNA capture sequence). For the miRNA capture sequence, a well-known oncomiR overexpressed in various cancers, miRNA-21, was chosen as a model target.40 As a result, aromatic bases of DNA wrapping around SWCNT surface through π−π bonding and anionic phosphate group improves solubility of SWCNT.41,42 Collectively, all of these contributes to the dispersion of SWCNT in aqueous solution, which is supported by AFM and TEM images of DNA-wrapping SWCNTs (Figure S1). In addition, the UV-visible absorption spectrum of DNA wrapped SWCNTs also showed the signal of DNA at about 260 nm (Figure S2). Note that the original properties of the SWCNTs would not be disrupted by such non-covalent attachment.43 Single-Particle Fluorescence Analysis for the Operation of DSN-Propelled RNA Walkers. The DSNpropelled RNA walking on SWCNT was next monitored by total internal reflection fluorescence microscopy (TIRF). To this end, a fluorophore (i.e., 6-FAM) labeled at the 3'-end of miRNA capture sequence serves as signal reporter. The RNA walking stand was first hybridized with miRNA capture probes on the SWCNT. Following, fluorophore release from SWCNTs was induced by DSN-mediated cleavage of the FAM-labeled DNA, leading to fluorescence loss of the focused single SWCNT under TIRF (Figure 2a). Initially, 62 fluorescent spots were observed in the imaging area of interest (Figure 2b, upper panel), followed by that the operation of the RNA walker made approximately 90 % spots diminished. In the absence of RNA walking strands, only 7% of the fluorescent spots yet diminished (4 out of 59, Figure 2 b, lower panel). In addition, we recorded a typical walking process in real time (Figure 2c, d). In the presence of RNA walking strands, a rapid fluorescence decrease (65% fluorescence loss) was monitored,

Figure 2. Single-particle fluorescence analysis for the operation of DSN-propelled RNA walkers using TIRF microscopy. (a) Schematic for the mechanism of an RNA walker moving on the SWCNT. (b) TIRF images for RNA walkers before (left) and after (right) adding DSN. (c, d) Representative timelapse images of single SWCNT in the presence (upper) and absence (lower) of RNA walking strands and their corresponding fluorescence intensity.

DSN-Propelled RNA Walkers for miRNA Detection. Given its powerful capability for amplified signal outputs, the RNA walker was exploited for miRNA detection. Its feasibility was first studied. As shown in Figure 3a, in the absence of target miRNA, the fluorescence of DNA probes was almost entirely quenched regardless of the presence or absence of DSN, which is attributed to the strong π-π stacking interaction between the ssDNA probe and SWCNT, as well as the high fluorescence quenching capability of SWCNTs.44 When target miRNA hybridized with miRNA capture domain (Figure 3b), the fluorescence of DNA probes was partially recovered, which arises from 1:1 binding event. However, the operation of RNA walker would produce numerous short FAM-labeled oligonucleotide fragments when incubating the SWCNT-based probes with target miRNA and DSN (Figure 3a). In our system, 700 FAM-labeled oligonucleotides were cut from single miRNA binding due to the RNA walker. As a result of the weak affinity of the short FAM-labeled oligonucleotide fragments to SWCNTs, strong fluorescence signal with a signal-tobackground ratio of ∼9 was observed (Figure S3), confirming that our RNA walker can be utilized for sensitive miRNA detection.

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Table 1. The comparison with other fluorescence methods for miRNA detection

Methods

Linear range

LOD

Selectivity

Ref.

Fluorescence detection

5 fM to 10 pM

1.67 fM

one-base difference

this work


10 pM to 1 nM

2.3 pM

one-base difference

55


0.5 pM to 500 pM

0.4 pM

one-base difference

56


1 pM to 10 nM

300 fM

one-base difference

57


100 pM to 100 nM

100 fM

four-base difference

45


100 fM to 1 nM

58 fM

one-base difference

17


1 pM to 10 nM

10 fM

one-base difference

36


25 fM to 1 pM

10 fM

one-base difference

46


0 to 0.75 nM

5 fM

────

49


10 fM to 10 pM

∼3.4 fM

one-base difference

37


────

2.6 fM

multiple-base difference

47


5 pM to 5 fM

2.1 fM

one-base difference

58


1.5 fM to 9 fM

1.5 fM

one-base difference

59


10 fM–100 pM

1 fM

multiple-base difference

48

To achieve the best assay performance, the amplification and sensing conditions including incubation time and the amount of used DSN were then optimized. In view of the stability of SWCNT-based probes, the incubation temperature of 40 °C was chosen. The concentration of SWCNT-based probes and RNA were fixed at 10 g/mL and 10 nM, respectively. In addition, the optimum reaction time was found to be 30 min (Figure S4). Seen from Figure S5, an optimal fluorescence intensity ratio (F/F0, where F0 and F are the fluorescence signals in the absence and the presence of miRNA, respectively) was observed when the amount of DSN was 0.1 U. Hence, 0.1 U DSN was used throughout subsequent experiments.

Figure 3. MiRNA detection with RNA walker. Schematic for stochastic RNA walkers moving on the SWCNT in the (a) presence and (b) absence of DSN and their fluorescence spectra. (c) Fluorescence spectra of RNA walkers in the presence of DSN in response to different concentrations of miRNA-21. (d) Histogram of fluorescence ratio (F/F0 − 1) as a function of the

concentration of miRNA, where F0 and F represent the FAM fluorescence intensity of SWCNT-based probes and DSN in the absence and presence of miRNA, respectively. Inset: linear response range of miRNA concentration. (e) Fluorescence ratio (F/F0 − 1) in response to different miRNAs targets. Error bars are standard deviations of three repetitive experiments.

The RNA walkers for quantitative analysis of miRNA was further investigated under the optimum conditions. As shown in Figure 3c, a dramatic fluorescence increase was observed with the increasing miRNA-21 concentration from 5 fM to 10 nM. Figure 3d illustrates the relationship between the fluorescence intensity ratio (F/F0 − 1) and different miRNA-21 concentrations, in which about 19-fold fluorescence enhancement is clearly observed at the concentration of 10 nM. Additionally, we found that the (F/F0 − 1) value possesses a linear correlation to the logarithm of miRNA-21 concentration ranging from 5 fM to 10 pM (Figure 3d, inset), spanning over four orders of magnitude. The calibration equation was lg (F/F0 − 1) = 6.3485 + 1.1512 lg C, with a correlation coefficient R2 = 0.993. The limit of detection (LOD) was estimated to be 1.67 fM according to 3σ rule, which was better or comparable to some previously reported fluorescence sensors for miRNA detection (Figure S6 and Table 1).36, 37, 45-50 The excellent sensitivity is mainly ascribed to the high fluorescence quenching capability of SWCNTs and signal amplification of RNA walkers. A significant challenge for miRNA analysis is the ability to distinguish miRNA with high homology, which is beneficial for understanding the biological functions of individual miRNA.51-53 miRNA-21 is reported as an antiapoptotic factor in cancer cells and shows overexpression in various cancers including breast, ovarian, lung, prostate, pancreas cancer, and glioblastomas.54 Figure 3e exhibits the comparison of fluorescence signals’ response toward

D


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Analytical Chemistry different miRNA targets. There was only a weak signal (F/F0 − 1) in the presence of one-base mismatched miRNA in contrast to the perfectly complementary target miRNA-21 (Figure 3e). Negligible fluorescence responses were observed for two-base mismatched or multiple-base mismatched miRNAs. Additionally, we also tested detection of miRNA-21 in the presence of an equivalent mixture of other miRNAs; the fluorescence ratio was almost the same as that in the presence of only miRNA-21. Moreover, an equivalent of target miRNA in complex systems (e.g., human serum) showed no obvious signal change, whereas serum without target miRNA displayed no obvious fluorescence signal (Figure 3e). These results suggest our RNA walker with high selectivity for miRNA analysis. The high specificity of RNA walker arises from good capability of DSN to discriminate perfectly from imperfectly matched DNA/RNA heteroduplexes. Stochastic RNA Walkers for Intracellular miRNA Imaging. Our nanomachine was then applied for intracellular miRNA-21 imaging by tailoring the design to facilitate the intracellular delivery of RNA walkers and DSN. Here, HeLa cells with high miRNA-21 expression profiles were selected as a model.60, 61 As illustrated in Figure 4a, Lipofectamine-2000 was used to encapsulate SWCNTbased probes and DSN, aiding for uptake of DSN by HeLa cells.62 Following, we investigated the cytotoxicity of nanomachine uptake to HeLa cells. The CKK-8 assay showed the complex of SWCNT-based probes and liposome-containing DSN with negligible side effects on the viability of HeLa cells within 24 h (Figure S7). The result revealed that our nanomachine can be used in living cells. The flow cytometry analysis was next carried out to demonstrate the fluorescence observation of RNA walker

Figure 4. Intracellular imaging of miRNA-21. (a) Schematic of RNA walkers for intracellular RNA imaging. (b) Imaging flow cytometry analysis of HeLa cells and (c) their relative fluorescence intensity. (d) Confocal laser microscopy images of miRNA-21 treated with RNA walkers. Scale bar: 10 m.

existing in the entire cell populations (Figure 4b). We found that our nanomachine and DSN were efficiently delivered into HeLa cells, showing significant fluorescence signal (Figure 4b, lower panel). In contrast, no fluorescence was observed from control group and a slightly fluorescence signal from that without DSN. The fluorescence intensity analysis in Figure 4c indicates that our RNA walkers perform walking in the living cells and thus produce enhanced fluorescence signal. To further confirm that our walker enables intracellular miRNA imaging, confocal fluorescence microscopy was conducted to monitor RNA imaging. As expected, the experimental group showed the brightest fluorescence signal, whereas those control groups (including control, random probe + DSN, probe + DSN + anti-miRNA AMO, and probe group) showed no or weak intracellular fluorescence signal (Figure 4d), which are well corresponding with the results of flow cytometry analysis. These results indicate that the enhanced signal is produced by miRNA-21 and our stochastic RNA walkers can be applied for intracellular miRNA imaging.

CONCLUSIONS In summary, we have developed an enzyme-propelled SWCNT-based RNA walker for intracellular RNA imaging. The movement of RNA walker on 1D SWCNT track transducing and amplifying biorecognition signals provides an important approach to construct biosensors. This walking machine possesses several distinctive traits for RNA analysis. First, DSN-mediated cleavage reaction allows the nanomachine for performing automatic and progressive walk on the SWCNT in the presence of target RNA. The high target-recycling kinetics results in amplified signal outputs. Second, DSN-powered RNA walker enables sensitive and specific detection of RNA with a LOD of 1.67 fM and with capability for one-base mismatch discrimination. Third, the machine and DSN can be effectively delivered to HeLa cells with the aid of transfection agent and achieve miRNA imaging in living cells. Finally, the rational design of RNA walkers in a programmable way offers new insight into expanding the application field of nanomachines to living cells.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. AFM and TEM images of DNA-wrapping SWCNTs (Figure S1), UV-visible absorption spectrum of DNA wrapped SWCNTs (Figure S2), relative fluorescence intensity for the SWCNT-based probe, probe + DSN, walker, and walker + DSN (Figure S3), time course of probe + DSN and probe + DSN + miRNA (Figure S4), the fluorescence spectra of RNA walkers upon addition of different amounts of DSN enzyme and their fluorescence signal E



ratio (Figure S5), fluorescence spectra of RNA walkers in the absence of DSN in response to different concentrations of miRNA-21 and linear response range of miRNA concentration (Figure S6), cytotoxicity analysis to HeLa cells of the complex of RNA walkers and liposomes by CCK-8 assay(Figure S7); the sequences of the involved oligonucleotides (Table S1) (PDF).

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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

ORCID Li Li: 0000-0001-8494-4997 Hao Pei: 0000-0002-6885-6708

Notes The authors declare no competing interest.

ACKNOWLEDGMENT This work was supported by the National Science Foundation of China (Grant No. 21722502) Shanghai Rising-Star Pro-gram (19QA1403000) and Shanghai Science and Technology Committee (STCSM) (Grant No. 18490740500).

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Stochastic RNA Walkers for Intracellular MicroRNA Imaging

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