Efficient and Reliable MicroRNA Imaging in Living Cells via a FRET

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A FRET-based Localized Hairpin DNA Cascade Amplifier Enables Rapid, Efficient, and Reliable MicroRNA Imaging in Living Cells Lu Liu, Qiming Rong, Guoliang Ke, Meng Zhang, Jin Li, Yingqian Li, Yongchun Liu, Mei Chen, and XiaoBing Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05778 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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Analytical Chemistry

A FRET-based Localized Hairpin DNA Cascade Amplifier Enables Rapid, Efficient, and Reliable MicroRNA Imaging in Living Cells Lu Liu,† Qiming Rong,† Guoliang Ke,†, * Meng Zhang,† Jin Li,† Yingqian Li,† Yongchun Liu,‡ Mei Chen,‡ Xiao-Bing Zhang †,* †

Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China ‡

College of Materials Science and Engineering, Hunan University, Changsha, 410082, China

*To whom correspondence should be addressed. E-mail: [email protected], [email protected] ABSTRACT: MicroRNAs (miRNAs) play critical roles in many biological processes and are vital biomarkers for disease diagnostics. Hence, it is of significance to develop miRNAs biosensors with fast response, high sensitivity and excellent reliability in living cells. As one kind of DNA molecular machines, DNA amplifiers are very promising for intracellular miRNAs imaging due to their nonenzymatic, isothermal working principle and excellent signal amplification ability. However, the practical application of current DNA amplifiers is still an issue because of their slow kinetic, unsatisfied efficiency and false positive signal. Herein, taking advantage of the spatial confinement effect on a three-dimensional (3D) finite DNA nanostructure, a FRET-based localized hairpin DNA cascade amplifier (termed localized-HDCA) is developed for the rapid, efficient, and reliable imaging of intracellular tumorrelated miRNA. The localized-HDCA system consists of two metastable hairpin DNAs (H1 and H2) localized on a DNA nanocube. Benefiting from the spatial confinement effect in the confined space of DNA nanocubes, not only the speed of miRNAs-triggered HDCA reaction was significantly accelerated (7 times faster), but also the reaction efficiency was greatly improved (2.6 times higher). In addition, the FRET-based 3D finite DNA nanocubes provide this localized-HDCA improved cell permeability and better nuclease resistance, as well as the ability to avoid false positive signal, which guarantee the reliable miRNAs imaging in living cells. With these advantages, this strategy is expected to be widely applied to the development of more efficient and robust DNA molecular machines for biomedical research and clinical diagnosis.

MicroRNAs (miRNAs) are a class of single-stranded, small (approximately 19-23 nucleotides), non-coding RNA that serve as key controllers of gene expression.1-3 In particular, the aberrant expression of miRNAs is closely associated with a large number of human diseases. Hence, miRNAs have been regarded as highly promising biomarkers in medical diagnosis3-5. Considering the low abundance of miRNAs in cells, the development of accurate and efficient amplification method is crucial for intracellular miRNAs imaging. Currently, enzymatic signal amplification methods, e.g. polymerase chain reaction (PCR)2, 6 and rolling circle amplification (RCA)7-10, have been explored for detecting rare miRNAs. However, the requirement of exogenous enzymes makes them not suitable for visualization of miRNAs inside living cells. 11 Thus, it is necessary to develop a non-enzymatic signal amplifier for visualization of intracellular miRNAs. As one kind of DNA molecular machines, DNA amplifiers are very promising for low-abundance biomarkers detection because of their excellent signal amplification ability.12-15 For example, a DNA amplifier named hairpin DNA cascade amplifier was developed for signal enhancement-based sensing of targets. More importantly, due to its nonenzymatic and isothermal features, DNA amplifiers are able to efficiently visualize the targets inside live cells.11 However, several pivotal issues need to be addressed before their real application of these DNA amplifiers.16-18 First, the reaction of traditional DNA amplifiers occurs through the random diffusion of DNA substrate for collision in a bulk solution, which makes the kinetic and efficiency quite low. Second, single-intensitybased detection system employed in current amplifiers may lead to false-positive signals due to the influence of concentration and local distribution of probes, environmental fluctuations, and unstable light sources. Simultaneously, the complex

environment in living cells would influence the stability of DNA probes. Therefore, it is significant to develop a rapid, efficient, and reliable DNA amplifier with not only improved kinetic and efficiency, but also minimized false-positive signal in the complex intracellular environment. Cells use spatial confinement effect to regulate and accelerate complex information-processing tasks within a sophisticated, and crowded intracellular environment.19-21 The spatial confinement effect could significantly accelerate the interactions among components that are closer together. Inspired by this phenomenon, several models have been demonstrated that confining successive reactants together in a compact space maintains high local concentrations of reagents and accelerates reactions.22-24 Among them, DNA nanostructures provide an ideal scaffold for spatial confinement study due to their addressable assembly of related components with nanometer precision.22, 25-32 For example, through the addressable assemble of enzyme cascades on a DNA nanostructure platform, we have significantly improved the speed and efficiency of enzyme cascade reaction.33-34 Similarly, this spatial confinement strategy on DNA nanostructure could also been applied in the reaction related to DNA. For example, Seelig et al. have developed a spatially localized molecular circuit on DNA origami for the fast and efficient DNA computing.29 Considering the key role of spatial confinement effect, we hypothesized that a spatially localized strategy could be a useful approach to develop fast and efficient DNA amplifiers for miRNA imaging. Moreover, different from the single-intensity-based detection system used in current DNA amplifiers, ratiometric-based readout is expected to avoid the false-positive signals and reduce the influence of system fluctuations.35 Herein, introducing the spatial confinement effect and FRET-based readout to DNA amplifiers, we developed the first FRET-based localized

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hairpin DNA cascade amplifier (termed localized-HDCA) for the rapid, efficient, and reliable intracellular microRNA imaging. As shown in Scheme 1, the localized-HDCA consists of two kinds of metastable hairpin DNA (H1 and H2) and a threedimensional (3D) DNA cubic structure. The H1 and H2 respectively modified with Cy3 and Cy5 were integrated into DNA nanocube through designed complementary strands. In the absence of a target, H1 and H2 remain intact, thus inducing low FRET efficiency. However, the presence of a target resulted in the formation of H1-H2 duplexes, switching FRET from the “off” to “on” state. At the same time, the target miRNA was released to trigger the formation of other H1-H2 duplexes, thus achieving the amplification detection of tumorrelated miRNA. Due to the spatial confinement effect in a confined space of DNA nanocubes, the reaction speed and efficiency of target-triggered HDCA reaction were significantly improved compared to traditional HDCA in bulk solution. In addition, the FRET-based 3D finite DNA nanocubes also provide localized-HDCA the ability to avoid false-positive signals through the employment of FRET readout and improved biostability. With these advantages, this localizedHDCA enables the rapid, efficient and reliable miRNAs imaging in living cells. Scheme 1. Schematic illustration of localized hairpin DNA cascade amplifier (localized-HDCA) for the rapid, efficient, and reliable miRNA biosensing in living cells. H2 Cy3

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MATERIALS AND METHODS Materials and Apparatus. All DNA sequences (Table S1) with high-performance liquid chromatography (HPLC) purification, were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). Other reagents which no specific explained were analytical grade, and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure water was obtained from Milli-Q system (Billerica, MA, USA). The spectrofluorometer for fluorescence measurements was Fluoromax-4 (HORIBA Jobin Yvon Inc., Edison, NJ). The AFM characterization of the sample was carried out on a Bruker Multimode V8 Scanning Probe Microscopy (Bruker, German). A FV1000 confocal laser-scanning microscope (Olympus, Tokyo, Japan) was used for the confocal fluorescence imaging studies. MTS assay was finished with a Synergy 2 Multi-Mode Microplate Reader (Bio-Tek, Winooski, VT). Preparation of localized-HDCA. DNA nanocube was selfassembled according to previous reports.36 In brief, equimolar amounts of Clip1, Clip2, Clip3 and Clip4 were combined in

1×TAE/Mg buffer (20 mM Tris, 2 mM EDTA, 12.5 mM MgCl2, pH 7.4). Samples were annealed with the following protocol: held at 95 ℃ for 5 minutes then 80 ℃ for 3 minutes, cooled to 60 ℃ (2 min/ ℃) and finally slowly cooled to 4 ℃ (3 min/ ℃). Then equimolar amounts of DNA nanocube, H1 and H2 were combined in 1×TAE/Mg buffer in 37 ℃ for 30 min. The probes were prepared for AFM characterization by depositing the samples onto a freshly cleaved mica. Electrophoresis Characterization. To characterize the self-assembly formation of localized-HDCA, native polyacrylamide gel electrophoresis (N-PAGE) was applied. The samples were run by 8% N-PAGE in 1×TAE/Mg buffer at 110 V and analyzed using a FLA-3000G image scanner (Fuji, Tokyo, Japan). To study the nuclease digestion of nanoprobes, different concentrations of DNase I were added to a 1 μM localized-HDCA nanoprobes and incubated for different times at 37 ℃. Then, 2 μL of loading buffer (6×) was mixed with 10 μL resultant nanoprobes, followed by 5% N-PAGE analysis. Fluorescence Measurementss. For verificationdetection of targets response in buffer solution, we added different concentration of targets (0, 0.5, 1, 5, 10, 15, 20, 30, 40, 50, 70 and 90 nM) to 200 μL of 1×TAE/Mg buffer containing 50 nM localized-HDCA nanoprobes. Then, after incubation at room temperature for 1.5 h, the fluorescence spectra of samples were measured a 200 μL quartz cuvette (the excitation wavelength was 525 nm, and emission wavelength were collected from 540 to 750 nm). To compare the ability to avoid false-positive signals of FRET-based localized-HDCA and molecular beacon-based nanoprobes, 50 nM of nanoprobes were incubated with same concentration of DNase I for different times (from 0 to 60 min) at 37 ℃ and analyzed by a fluorescence image scanner. Cell Culture. Human embryonic kidney cells 293 (HEK293), Human cervix carcinoma cell line (HeLa) and human breast adenocarcinoma cell line (MCF-7) were obtained from our lab. 1 % penicillin-streptomycin (PS, 10 000 IU penicillin and 10 000 μg/mL streptomycin, Multicell) and 10 % fetal bovine serum (FBS) were added into 1640 medium (GIBCO) for cells culture. Cytotoxicity. Cytotoxicity of the localized-HDCA nanoprobe was assessed with a standard MTS assay. HEK293 cells, HeLa cells and MCF-7 cells were cultured in 96-well plates, followed by the incubation at 37 ℃ in 5 % CO2 for 24 h. Different concentrations of localized-HDCA nanoprobes (0 and 200 nM) were added to each well and incubated for different time (6, 12 and 36 h, respectively). Subsequently, after washing with DPBS for three times, the cells were mixed with 10 μL of MTS and incubated at 37 ℃ for 2 h. Finally, the absorbance at 490 nm of samples was measured using a multimode microplate. Confocal Fluorescence Imaging. In the cell imaging experiments, HEK293, HeLa and MCF-7 cells were cultured at 37 ℃ with 5 % CO2 for 24 h. The culture medium contains 10 % fetal bovine serum and 1% penicillin-streptomycin. Next, the cells were washed three times with DPBS buffer and incubated with respective probes at 37 ℃ for another 6 h. After washing the cells with DPBS for three times, confocal fluorescence imaging of different kinds of cells were carried out on a Nikon ultra-high resolution spectroscopic confocal microscope.

RESULTS AND DISCUSSION Preparation and Characterization of localized-HDCA. The localized-HDCA consists of two kinds of metastable hair-


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Analytical Chemistry pin DNA (H1 and H2) and a 3D DNA cubic structure (Scheme 1). The H1 and H2 modified with Cy3 and Cy5 respectively were integrated into DNA nanocube through designed sequence-complementary DNA hybridization (Table S1). In the absence of a target, since the hybridization reaction of H1 and H2 was effectively restrained, H1 and H2 remained intact. The donor (Cy3) and receptor (Cy5) were spatially separated to exhibit the FRET “off” signal. However, the presence of target miRNA resulted in the formation of H1-H2 duplexes, switching FRET from “off” to “on” state. Moreover, after triggering H1 and H2 to form a stable hybridized duplex, target miRNA was released from H1-H2 to catalyze the formation of other H1-H2 duplexes, thus achieving efficient amplification of signal. We first investigate the formation of DNA nanocubes through native polyacrylamide gel electrophoresis. With the stepwise hybridization of DNA strands, DNA nanocubes were successfully assembled with high yield, confirmed by the gradual reduction of electrophoretic mobility attributed to the increased molecular mass (Figure S1). Subsequently, localized-HDCA probes were prepared by assembly of DNA nanocubes with H1 and H2. As shown in Figure 1b, after mixed with H1 and H2, the band of DNA nanocubes turned to be slower, indicating the successful formation of localizedHDCA. Moreover, the localized-HDCA was also directly characterized by atomic force microscopy (AFM), which demonstrates that our localized-HDCA was appeared to be monodisperse (Figure 1a). Compared with previous DNA nanowires that are difficult to finite control on size, this DNA nanocubes-based localized-HDCA has finite structure and monodisperse size, which could exhibit better biological behavior. 37

only Cy3 fluorescence in the absence of target and demonstrated a FRET signal proportional to target concentration due to the opening of H1 and H2 and corresponding close proximity between Cy3 and Cy5 (Figure S3). Moreover, the catalytic amplification of localized-HDCA triggered by target was further confirmed by comparing with a non-amplificatory probe. As shown in Figure S4a-b and Table S1, the H2 strand in a localized-HDCA was replaced by H2-nonamplification strand in a non-amplificatory probe. With this design, the segment for releasing the target was cut off to avoid catalytic amplification process. As a result, the FRET signal response of nonamplificatory probe significantly decreased comparing with that of localized-HDCA (Figure S4c), indicating the feasibility of target triggered amplification of localized-HDCA. Kinetic Analysis of Accelerated HDCA in localizedHDCA. To study the reaction kinetics of probes, timedependent fluorescence analysis was carried out. To verify the effect of localized-HDCA, a control group, free HDCA without DNA nanocubes (Figure S5a), was also tested. After mixing these two kinds of probes with the same concentration of target respectively, the time-dependent fluorescence spectra were measured every 5 minutes. As shown in Figure 2a, compared with free HDCA, localized-HDCA exhibited much faster fluorescence response. Comparing the slopes of timedependent fluorescence curve, the reaction rate of localizedHDCA is about 7 times faster than that of free HDCA, which indicating the significant improvement on the reaction speed. Meanwhile, the FRET change of localized-HDCA was about 2.6 times higher than that of free HDCA, indicating the higher reaction efficiency of localized-HDCA. To explain the mechanism of the fast kinetic and higher efficiency of localized-HDCA, the collision theory was introduced to analyze the reaction process. The equation of V=1/cN (where c is the concentration of H1 or H2, and N is Avogadro constant) was employed to connect the volume and the concentration of probes. For free HDCA probe with 50 nM H1 and H2, the volume of a sphere containing both H1 and H2 molecules was calculated to be 3.32×10-17 L with a radius of 199 nm (Figure 2b). In the localized-HDCA system, the distance between H1 and H2 was about 20.4 nm (60 bp) including the anchoring segment and diagonal line length of DNA nanocube, thus the local concentrations of H1 and H2 in (a)

Figure 1. (a) Atomic force microscope characterization of the probes. The scale bar is 100 nm. (b) 8% native-PAGE for assembly of localized-HDCA. Lane 1: DNA nanocube; Lane 2: DNA nanocube with H1; Lane 3: DNA nanocube with H2; Lane 4: DNA nanocube with H1 and H2 (localized-HDCA). Feasibility of Target Triggered Amplification of localized-HDCA. The catalytic feasibility of the hairpin DNA cascade amplifier was first verified in homogeneous solution with free H1 and H2 via 8% PAGE analysis. As shown in Figure S2, the presence of target DNA resulted in the formation of H1-H2 duplexes with lower mobility, while the bands representing H1 and H2 disappeared (lanes 5, 6 and 7). The response of probes to target strand was further investigated using fluorescence experiments. The localized-HDCA showed

(b) 46.8 μM 3.55×10-20 L

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Figure 2. (a) Time-dependent fluorescence spectra of different kinds of HDCA probes in response to same concentration of target. (b) Comparison of the reaction area and local concentration of H1 and H2 for free HDCA and localized-HDCA.


Analytical Chemistry

Figure 3. (a) Fluorescence spectra of different concentrations of target. (b) The relationship between the ratio of acceptor to donor emission (FA/FD) and target concentration. (c) The specificity of localized-HDCA. The data error bars indicate means ± SD (n=3). localized-HDCA was calculated as 46.8 μM, which is 936-fold as compared with the situation in free probes.31-32 Since the collision frequency is proportional to the reactants concentration, the collision frequency between H1 and H2 in localizedHDCA was significantly enhanced, resulting in the accelerated reaction speed and improved reaction efficiency.29, 31 In Vitro Fluorescence Response of localized-HDCA. In vitro target binding studies of the probes were performed by the addition of different concentrations of target. As shown in Figure 3a, when target concentration increases, the fluorescence emission intensity of Cy3 at 565 nm decreased rapidly, with the increase of Cy5 emission at 662 nm. The fluorescence emission ratio of acceptor to donor (FA/FD) was utilized to represent FRET signal. As shown in Figure 3b, a gradual increase in FRET signal was observed with the increase of the target concentration. Moreover, the FA/FD signal of localizedHDCA was much higher than that of free HDCA (Figure S5) under the same incubation conditions, indicating that the localized-HDCA could achieve more effective signal amplification and higher detection sensitivity. The detection selectivity of localized-HDCA was also investigated. As shown in Figure 3c, compared with target, random DNA showed very low fluorescence change, which was in accordance with the blank sample. In addition, we also replaced H2 with a hairpin strand with random sequence (H2random). The result showed that the FA/FD signal of H2random (non-probe) with target was only about 8% of that of localized-HDCA (Figure 3c). These results demonstrated the excellent selectivity of localized-HDCA. Ability of localized-HDCA to Avoid False-positive Signals. A key problem that limits the real application of DNA amplifiers comes from the false-positive signals in complex biological systems. On the one hand, endogenous nuclease digistion could trigger DNA probes to be out of action in living cells. On the other hand, current DNA amplifiers based on single-intensity-based detection system are easily affected by experimental conditions such as the concentration of probes and unstable light sources. With improved stability and the employment of FRET readout, our localized-HDCA is expected to address the issue of false-positive signals. As a proof-of-concept, the interaction of localized-HDCA with DNase I was first investigated. As shown in Figure 4a, after treatment with 0.5 U/mL DNase I (a higher concentration than that in living cells), the localized-HDCA showed almost no degradation, indicating that DNA nanocubes could improve the nuclease resistance of the oligonucleotides on the nanostrcture.38 Although the improved stability was also reported in previous nanomaterials-based DNA probes, the FRET readout of localized-HDCA further minimize false-

positive signals comparing to current single-intensity-based detection system. To better demonstrate this concept, FRET readout-based localized-HDCA was compared with a singleintensity readout-based molecular beacon. The molecular beacon (H2-FQ in Table S1) was also anchored on the same DNA nanocube, but only showed the intensity of a single fluorescent dye upon the presence of target. As shown in Figure S6, an obvious increase of fluorescence intensity of molecular beacon probes was detected upon the addition of DNase I, indicating that single-intensity readout-based molecular beacon suffers from the problem of false-positive signals. In contrast, even the localized-HDCA was completely digested by excess DNase I, the FA/FD signal showed negligible change (Figure 4b). Therefore, our localized-HDCA nanoprobes were able to avoid false-positive signals, which is highly important for intracellular accurate bioimaging. FRET “OFF”

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Figure 4. Ability of localized-HDCA to avoid false-positive signals. (a) Gel characterization for the degradation of localized-HDCA nanoprobe by different concentrations of DNase I. (b) The fluorescence response of probes treated with different concentrations of DNase I. Live Cell Imaging of miR-21 via localized-HDCA. To investigate the he biocompatibility of localized-HDCA probes, a standard MTS assay was performed on HEK293 cells (human embryonic kidney 293 cell line), HeLa cells (human cervix carcinoma cell line) and MCF-7 cells (human breast adenocarcinoma cell line), respectively. In this case, 200 nM localizedHDCA probes were added into the culture medium of these cells. Figure S7 indicated that cells could retain above 90% viability after the treatment of the probes. The results proved that our probes were safe to the cells. We next applied the localized-HDCA for imaging of miRNA-21 in living cells. To better demonstrate the merit of localized-HDCA to internalization into cells, nude H1H2 probes were incubated with HEK293 cells, HeLa cells and MCF-7 cells, respectively. As shown in Figure S10, negligible fluo-


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Analytical Chemistry rescence or FRET signal was observed in the cells. This suggested that the nude HDCA is not suitable for intracellular miRNA imaging due to its poor permeability and low biostability. In contrast, the fluorescence signals of cells treated with localized-HDCA were significantly increased, suggesting the successful cell uptake of our probes (Figure S8). To better investigate the intracellular localization of localized-HDCA, the z-stack images of HEK293 cells treated with localizedHDCA were carried out. The result showed that the localizedHDCA exhibited excellent self-delivery38-39 into cells without any transfection agent (Figure S9). These results proved that localized-HDCA can enter living cells due to its structure rigidity, which is in accordance with previous DNA nanostructure-related reports.37 Having demonstrating localized-HDCA can efficiently enter cells, we further studied the ability of localized-HDCA to visualize specific intracellular targets. Benefiting from its nonenzymatic and isothermal features, the localized-HDCA was applied for in situ detection of miRNA-21 in live cancer cells. According to previous reports, miRNA-21 is overexpressed in HeLa cells and MCF-7 cells, but is absent in HEK293 cell40-42. Therefore, same amount of probes (200 nM) was incubated with these three kinds of cells for 6 h, followed by confocal fluorescence imaging. The result showed that the FRET signal for miRNA-21 in HeLa cells and MCF-7 cells was significant, while negligible FRET signal could be detected in HEK293 cells (Figure 5). It suggested that our localized-HDCA not only showed excellent self-delivery property, but also was able to distinguish different cell lines with distinct miRNA expression levels. Although the abundance of miRNAs in living cell is very low, localized-HDCA can achieve the obvious imaging of miRNAs due to the significant amplification effect of HDCA. The results indicated that our localized-HDCA could be applied for rapid, efficient, and reliable imaging miRNA in living cells with ability of self-delivery, accelerated speed and signal amplification.

be observed. The results demonstrated that only true target can trigger the response of localized-HDCA, thus efficiently guaranteeing the reliable miRNA imaging in living cells.

CONCLUSION In summary, we have developed a FRET-based localizedHDCA for rapid, efficient, and reliable imaging of intracellular tumor-related miRNA. The localized-HDCA can be conveniently synthesized via simple hybridization of DNA nanocubes with respective FRET-based DNA amplifier elements. Compared to previous DNA amplifiers, our 3D finite DNA nanocubes-based FRET localized-HDCA exhibits its own remarkable advantages. First, due to the spatial confinement effect on DNA nanocubes scaffold, localized-HDCA can significantly improve the speed and efficiency of signal amplification for more rapid and efficient DNA amplifiers. Second, due to the “off-on” FRET signal readout strategy, the localized-HDCA efficiently avoid the generation of false-positive signals, thus ensuring the reliable miRNA imaging. Third, the 3D DNA nanocubes provide improved cell permeability and better biostability, which are important for keeping the performances of DNA probes. With these advantages, this new strategy could be applied to the imaging of other intracellular targets of interest including small molecule metabolites and proteins. We believe that these novel DNA molecular machines would open the opportunity for better understanding the functions of biomolecules in a vast range of biological processes.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Additional characterization data of gel electrophoresis, fluorescence spectra, laser scanning confocal microscopy tests, and cell viability assay; and Table S1, list of oligonucleotides sequences used in this study. (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], * E-mail: [email protected] Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

Figure 5. Fluorescence image of miRNA-21 in HEK293, HeLa and MCF-7 cells by localized-HDCA. Scale bars are 20 μm. The specificity of localized-HDCA for the intracellular miRNA-21 imaging was also demonstrated by incubating HeLa cells and MCF-7 cells with probe-r (H2 was replaced with a random sequence), which would not produce FRET signal upon miRNA-21. As shown in Figure S11, obvious Cy3 fluorescence were observed but almost no FRET signals could

This work was supported by National Natural Science Foundation of China (Grants 21705038, 21890744, 21705037, 21521063) and the Natural Science Foundation of Hunan Province (2018JJ3029, 2018JJ3092).

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Page 8 of 8

For TOC only H2

H1

FRET “OFF”

miRNA

Nucleus

FRET “ON”


8

Efficient and Reliable MicroRNA Imaging in Living Cells via a FRET

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