A NIR Light-Gated DNA Nanodevice for Spatiotemporally Controlled

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A NIR Light-Gated DNA Nanodevice for Spatiotemporally Controlled Imaging of MicroRNA in Cells and Animals Jian Zhao, Hongqian Chu, Ya Zhao, Yi Lu, and Lele Li J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b01931 • Publication Date (Web): 31 Mar 2019 Downloaded from http://pubs.acs.org on March 31, 2019

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Journal of the American Chemical Society

A NIR Light-Gated DNA Nanodevice for Spatiotemporally Controlled Imaging of MicroRNA in Cells and Animals Jian Zhao,† Hongqian Chu,† Ya Zhao,† Yi Lu,§ and Lele Li,*,†,‡ †CAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China ‡Center

of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, China §Department

of Chemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States

Supporting Information ABSTRACT: Nanodevices have potential as intelligent sensing systems for detection of microRNAs (miRNAs) in living cells. However, the resolution offered by “always active” nanodevices is often insufficient to manipulate microRNAs sensing with high spatiotemporal control. In this work, using DNA nanotechnology we constructed an activatable DNA nanodevice programmed to detect miRNAs in vitro and in vivo with the high spatial and temporal precision of NIR light. Our nanodevice is functionalized on the surface of upconversion nanoparticles (UCNPs) with a rationally designed DNA beacon that displays UV light-activatable miRNA sensing activity. The UCNPs absorb deep-tissue penetrable NIR light and emit high-energy UV light locally, which serves as transducers to operate the nanodevice in the NIR window. The nanodevice can naturally enter cells and enable remote regulation of its fluorescent imaging activity for miRNAs in living cells by NIR light illumination in chosen place and time. Furthermore, we demonstrate that the nanodevice can be expanded to activatable imaging of intratumoral miRNAs in living mice. This work illustrates the potential of DNA nanodevices for miRNA detection with high spatiotemporal resolution, which could expand the toolbox of technologies for precise biological and medical analysis.

INTRODUCTION DNA molecules have emerged as excellent substrates for the design of molecular machines that have the potential to sense external signals, actuate and execute highly complex tasks.1-3 Combined with their ready cellular internalization, DNA-based nanodevice represents an unparalleled opportunity to interface with biology and has been used as biosensing and imaging probes4-8 as well as cargo delivery vehicles.9,10 In particular, well-defined DNA nanodevices or nanoprobes have been designed for specific microRNAs (miRNAs) monitoring and detection in living cells,11-23 which could provide valuable information for biological study, medical diagnosis, and therapy. Despite of the progress made, most of the sensing probes are “always active” and work through the passive probe–miRNAs interaction with little temporal control of their imaging activity,11-20 thus could recognize and respond to targets encountered in transit, leading to lack of detection accuracy. Construction of engineered nanodevices for miRNA sensing and imaging with high spatial-temporal resolution is still a challenge. Light has emerged as a powerful tool for modulating biological functions both spatially and temporally because it can be delivered with high precision regarding time, space, dose and wavelength.24-26 Many strategies have

been developed to incorporate photolabile or photoswitchable moieties in nucleic acids or proteins for light-mediated control of cellular functions,27,28 therapeutics,29,30 or gene expression.31-34 Recently, lightcontrolled DNA nanoprobes have been developed for sensing and imaging of metal ions,35-38 small molecules,39,40 and miRNA22,23 in living cells. However, the requirement of ultraviolet (UV) or blue light in these systems22,23,27-37 is a significant limitation due to associated poor tissue penetration and phototoxicity. Moreover, due to these limitations, light-activated nanodevices serving as spatially- and temporally-controlled probes for miRNA imaging in vivo have—to our knowledge—not yet been reported. To address this unmet need, here we report the construction of a novel DNA nanodevice that combines a rationally designed photoactivatable DNA probe with photon upconversion nanotechnology to enable NIR light-activated spatiotemporal control of the miRNA imaging in living cells and in animals. The design allows the remote activation of the DNA probe with NIR light, which has much deeper tissue penetration than UV and visible lights, and is less phototoxic to cells. We chose miR-21, an important diagnostic marker of cancers, as the target miRNA to demonstrate the proposed design.

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Figure 1. Working principle of the activatable DNA nanodevice for NIR light-controlled miRNA sensing and imaging. (a) Schematic illustration of the design features of the DNA molecular beacon. (b) Schematic of DNA nanodevice for NIR light activated miRNA sensing in vivo.

Shown in Figure 1 is the principle by which to design the NIR light-activatable nanodevice for miRNA sensing. We first designed a UV light responsive DNA probe (PBc) through installation of a photocleavable (PC) bond into the hairpin loop of a molecular beacon, which would temporarily favor forming beacon structure and render the functionally inert to recognize the target miRNA. The PBc was labeled with fluorophore Cy5 and black hole quencher (BHQ2) at the opposite ends, respectively, to yield a low fluorescence background by Förster resonance energy transfer (FRET) prior to sensing. Upon UV light irradiation, the photolysis of the PC bond will shift the initial equilibrium to enable dose-dependent displacement of the quencher-labeled strand in the cleavage PBc with target miRNA, followed by significant fluorescent signal increase. Moreover, this designed DNA probe was equipped on upconversion nanoparticles (UCNPs) that acted as transducers to convert low-energy NIR light to high-energy UV light locally for the remote control over the activity of the DNA probe in the biological window. UCNPs processes photoconversion capability to absorb NIR light and emit UV and visible light through the sequential absorption of multi lowenergy photons.41-46 This unique property of UCNPs have been applied to drive photochemical reactions for various applications.41-46 For example, most recently, UCNPs has been used for NIR light mediated optogenetics to stimulate deep brain neurons.42 We have demonstrated NIR-activated imaging of ATP40 and metal ions38 in living cells and zebrafish based on integration of UCNPs with aptamer and DNAzymes, respectively. Yet no attention has ever been paid to the UCNPs-mediated, NIRcontrolled miRNA imaging in vitro and in vivo. RESULTS AND DISCUSSION Programming of DNA evaluated the feasibility of the

nanodevice.

We

first

Figure 2. Evaluation of the PBc probe for its UV light activated miRNA sensing performance in solution. (a) Fluorescence spectra of the PBc and Bc probes responding to 20 nM miR-21 with and without 365 nm light irradiation. (b) Fluorescence spectra of the PBc probe in the presence of 20 nM miR-21 with increased doses of 365 nm light irradiation. The fluorescence was measured immediately after light irradiation. (c) Fluorescence response of PBc as a function of miR-21 concentration with or without 365 nm light irradiation. (d) Fluorescence response of PBc to different miRNAs (20 nM) under 365 nm light irradiation. Data are represented as means ± s.d. (n = 3).

designed PBc as a photoactivatable sensing probe to synthetic target in a buffer. As shown in Figure 2a, without light irradiation, the fluorescence signal of PBc remained at the background level in the presence of miRNA target. In contrast, there is a significant enhancement of fluorescent signals when PBc is switched on by UV light irradiation (5 mW/cm2, 5 min) and subsequent addition of miR-21. These results indicated that the biosensing function of the PBc could be activated with an applied light illumination. Importantly, upon UV light irradiation, no significant fluorescence increase was seen for PBc without addition of miR-21 (Figure S1). As a positive control, the already cleaved DNA duplexes of the same sequence as PBc showed a sensitive miR-21-induced fluorescence response (Figure S2). As a negative control, the DNA probe (Bc) that possess the same sequence as PBc but without PC bond within its loop showed no obvious change in fluorescence intensity after the addition of target with or without photoactivation (Figure 2a, Figure S3), indicating that the light-triggered cleavages of the PC group was crucial for construction of the photoactivatable probe. The sensing performance of the PBc shows irradiation time-dependent activation: the longer the light irradiation, the more active probes were present to bind to target miRNA, resulting in higher fluorescence (Figure 2b), which peaked at 5 min of irradiation (Figure S4). Agarose gel electrophoresis was performed to confirm that ∼97% of the PBc can be photoactivated after 5 min of irradiation (Figure S5). The activated PBc showed a gradual increase in its fluorescence intensity according to increased concentrations of the target (Figure 2c). In contrast,


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Journal of the American Chemical Society minimum fluorescence change was observed for the nonirradiated PBc upon increasing miR-21 concentration. The selectivity of the PBc probe toward miR-21 over other miRNAs was also tested. As shown in Figure 2d and Figure S6, under light irradiation, fluorescence intensity induced by miR-21 was significantly higher than that caused by other miRNAs, suggesting that the specificity of the probe was high enough to discriminate between different miRNAs.

free cells. Strikingly, when cells were exposed to PBc-UN, significant increase of fluorescence signal was observed (Figure S12), suggesting that the DNA nanodevice could self-deliver into cells without using transfection agents. The time-dependent colocalization study showed that the nanodevice accumulates inside endo/lysosomes at the early stage of cellular uptake and then was able to escape from the endo/lysosome entrapment (Figure S13). This endosomal escape was further confirmed by a Z-stack imaging analysis (Figure S14).The results are consistent with the endosome-disrupting functionality of poly-lysine on the surface of nanoparticles, a cationic polymer used widely for gene delivery.47

Figure 3. Characterization of the PBc-UN. (a) EDS line scan profiles of a single core-shell structured UCNP along the arrowed line shown in HAADF-STEM image (inset). Scale bar: 20 nm. (b) Upconversion luminescence spectra of UCNPs and PBc-UN under excitation at 980 nm, respectively.

To establish NIR-to-UV transducers with high efficiency, we next performed epitaxial growth of optically insert NaGdF4 layer onto the as-prepared NaGdF4:70%Yb,1%Tm core by a thermal decomposition approach. Transmission electron microscopy (TEM) showed that the epitaxial growth led to the nanoparticles with size of 38.4 nm and shell of ∼5 nm (Figure S7). The controlled synthesis was verified by energy-dispersive Xray spectroscopy (EDS) line scan analysis of single UCNP (Figure 3a) and element mapping (Figure S8). Highresolution TEM imaging further confirmed the lattice structure of hexagonal NaGdF4 (Figure S9). The core-shell design could avoid surface-induced quenching of upconversion luminescence (UCL), leading to a significant enhancement of UCL (Figure S10). The obtained UCNPs were then coated with a cationic polymer (poly-lysine) for loading with PBc through electrostatic interactions. The assembly of PBc on the surface of UCNPs was confirmed by dynamic-lightscattering analysis and zeta potential measurement (Figure S11). TEM showed that the obtained PBc-UN are still monodispersed (Figure S9). The loading content of PBc was measured to be 30 probes per particle. The emission profile of the PBc-UN under excitation at 980 nm display characteristic emission bands of Tm3+ centered at UV (346 nm and 363 nm) and visible blue (453 nm and 478 nm) range (Figures 3). The upconverted UV emission matches the wavelength of light used for the miRNA sensing activation. NIR light-triggered DNA nanodevice for remotely controlled miRNA imaging in live cells. To evaluate the cellular uptake, HeLa cells were treated with only Cy5-labeled PBc or PBc-UN for 2 h. Cells treated with PBc exhibited little uptake due to the electrostatic cell membrane barrier, with similar fluorescence levels as that of

Figure 4. NIR light activated miR-21 imaging in live cells. (a) Confocal fluorescence images of HeLa cells treated with PBcUN with and without NIR irradiation. Scale bar: 10 μm. (b) Flow cytometry showing the miR-21 sensing activity of PBcUN and Bc-UN with and without NIR activation. (c) Quantification of the flow cytometric data in (b). Data are represented as means ± s.d. (n = 3).

We next turned our attention to NIR light-controlled activation of PBc-UN for miR-21 sensing in live cells. The fluorescent response of PBc-UN in HeLa cells with and without NIR light illumination (ON and OFF states, respectively) was compared using confocal laser scanning microscopy. HeLa cells were treated with PBc-UN for 2 h, and subsequently irradiated with a 980 nm NIR light (1.2 W/cm2). NIR-activation of PBc-UN is well-tolerated by the cells, as confirmed by the MTT viability assay (Figure S15). As shown in Figure 4a, without NIR irradiation, application of PBc-UN to HeLa cells resulted in very weak cellular fluorescence signals. In contrast, illumination with a 980 nm laser resulted in a significantly higher intracellular fluorescent signals, indicating that the sensing probe remained silent until photoactivation. A control experiment using the Bc-UN (UCNPs loaded with the inactive Bc probes) with no photoactivatable property revealed minimum change of intracellular fluorescence signals upon NIR irradiation (Figure S16). To further



verify these results from confocal microscopy, we quantified the intracellular signaling of the nanodevice using flow cytometry, which is a standard tool for the quantification of large population of cells and could eliminate variations observed with microscopy.21 The results indicated that PBc-UN provides a 3.2-fold increase in fluorescence intensity following NIR irradiation, while photoactivation of Bc-UN results in a similar cellular fluorescence intensity (Figure 4b and c). We then evaluate the efficiency of the system by estimating the intracellular saturation signal with only Cy5-labeled PBcUN (without labeling with quencher BHQ2). The results indicated that 27% signal was recovered upon the NIRactivated miR-21 binding (Figure S17). Together, these results strongly validate that spatially restricted NIR light can temporally control miRNAs sensing and imaging in living cells. In our design, we hypothesized that the light-triggered cleavage of the PC bond in PBc probe will enable the binding of the cleaved strand with miR-21 to form stable complexes. Since the formation of stable complexes of miR-21 with its complementary sequence is known to downregulate the intracellular levels of Bcl-2 messenger RNA (mRNA) and Bcl-2 protein,48,49 we could evaluate the binding of the cleaved probe with miR-21 by investigating the levels of Bcl-2 mRNA and protein. The qRT-PCR data and western blot analysis showed that NIR-irradiation of the PBc-UN in the cells indeed decrease the levels of Bcl-2 mRNA and protein (Figure S18). In contrast, nonirradiated PBc-UN and irradiated Bc-UN had no effect on the cellular expression of Bcl-2 mRNA and protein. Taken together, these results directly confirm that the observed fluorescence response for the PBc-UN was due to the NIR-activated binding of PBc probe with miR-21 in the cytosol. MiRNAs expression is dynamic, and expression of the same miRNA varied at different stages of tumorigenesis. Detection of fluctuations of miRNA level with DNA nanodevices has potential in diagnostic and therapeutic applications. Here, we evaluated the capability of the NIR light-activatable nanodevice to detect changes in concentrations of miR-21 in cells. It has been reported that miR-21 inhibitor, a modified single-stranded RNA molecule, could selectively bind and decrease intracellular miR-21 concentration, and that miR-21 mimic, a double-stranded RNAs mimicking miR-21, had the opposite effect.50 Therefore, HeLa cells were first treated with miR-21 inhibitor or mimic to regulate miR-21 levels. Then, the intracellular miR-21 levels for the untreated, inhibitor or mimic treated cells were determined with photoactivatable PBc-UN. As shown in Figure 5a, a distinguishable decreased fluorescence signals in the inhibitor treated cells but increased fluorescence signals in the mimic-treated cells could be observed compared with those in the untreated cells. Quantification with flow cytometry indicated that the fluorescence signal in the cells pre-treated with inhibitor was 1.9-fold lower than that in untreated cells (Figure 5b and 5c). In contrast, the fluorescence intensity in the mimic-treated cells was 2.5-

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fold higher than that of untreated cells (Figure 5b and 5c). Furthermore, this quantification data were consistent with the data obtained via conventional qRT-PCR technique (Figure 5c, Figure S19), confirming that the sensing results of the nanodevice were correlated very well with the intracellular miRNAs levels.

Figure 5. NIR light activated monitoring of the intracellular miR-21 fluctuations. (a) Confocal fluorescence images of HeLa cells pretreated with miR-21 inhibitor or miR-21 mimic, followed by treatment with PBc-UN and NIR light activation. Scale bar: 10 μm. (b) Flow cytometric quantification of the cells from (a). (c) Quantification of the relative intracellular miR-21 expression level through photoactivatable PBc-UN and conventional qRT-PCR technique, respectively. Data are represented as means ± s.d. (n = 3).

Next, we investigated the potential of PBc-UN for NIRactivated quantitative measurement of miR-21 in various types of cell lines. Two positive cell lines (MCF-7, HeLa) and one negative cell line (HEK293T), which display different expression levels of miR-21,51 were selected for miRNA detection with NIR-activatable PBc-UN. Cellular internalization study with only Cy5-labeled PBc-UN showed that the nanodevice exhibited similar cellular uptake capability for the three types of cell lines (Figure S20). As shown in Figure 6a and b, varying fluorescence signals were observed in these cells treated with PBc-UN followed by NIR light activation, with MCF-7 cells produced the strongest fluorescence signals whereas HEK293T cells possessed the lowest fluorescence intensity. The results were consistent with the reported expression levels of miR-21 in the three types of cell lines.51 Furthermore, the quantitative results were correlated well with the relative expressions of miR-21 measured by qRTPCR (Figure 6c, Figure S21).


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Journal of the American Chemical Society In another control study, five nucleotides in PBc probe are mutated to inhibit its specific miR-21 recognition (the resulting probe is named PBc(m)). NIR-activated fluorescence increase in vitro and in vivo was not observed for PBc(m)-UN (UCNPs loaded with the inactive PBc(m) probes) (Figure S23), eliminating any artifact due to nonspecific degradation of DNA. Similar to the cellular data, a specific decrease of Bcl-2 mRNA and protein levels in the tumor was observed only from the mice treated with PBc-UN and NIR irradiation, but not from the group treated with nonirradiated PBc-UN or irradiated Bc-UN (Figure S24). Consequently, it is conceivable that PBc-UN allows for NIR-triggered miRNA imaging in vivo.

Figure 6. NIR light activated analysis of miR-21 expression in different cell lines. (a) Confocal fluorescence images of MCF7 cells, HeLa cells, and HEK293T cells treated with PBc-UN and subsequent NIR activation. Scale bar: 10 μm. (b) Flow cytometric quantification of the cells from (a). (c) Quantification of the relative miR-21 expression in different cell lines via photoactivatable PBc-UN and qRT-PCR technique, respectively. Data are represented as means ± s.d. (n = 3).

NIR light-gated DNA nanodevice for spatiotemporally controlled miRNA imaging in vivo. Finally, we envisioned that PBc-UN could find utility in vivo wherein deep-tissue penetrable NIR light could guide spatially resolved photoactivation of PBc-UN for miRNA sensing in tumors. We first examined their intratumoral sensing and imaging performance upon direct injection at the tumor site. Nude mice bearing subcutaneous HeLa xenograft tumors (diameters ∼6–8 mm) on the left back were treated with PBc-UN or Bc-UN through intratumoral injection, followed by photoactivation at the tumor site with a NIR laser illumination (980nm, 1.2 W/cm2). Then, they were sent for quantitative wholebody fluorescence imaging with an in vivo imaging system. The mice injected with PBc-UN showed a significant fluorescence signal increase at the tumor site at different time points after photoactivation (Figure 7a). In contrast, no obvious fluorescence enhancement was observed for the mice treated with PBc-UN but without NIR irradiation. As shown in Figure 7b, quantification indicated that the treatment with PBc-UN and NIR irradiation led to 1.88- and 1.67-fold higher intratumoral fluorescence than that treated with PBc-UN but no illumination at 2 and 4 h, respectively. As a control, BcUN showed no obvious change of fluorescence at the tumor site with and without photoactivation (Figure S22).

Figure 7. NIR light activated miRNA imaging in tumor when DNA nanodevices were delivered by intratumoral injection. (a) Whole-body fluorescence imaging of HeLa tumor-bearing mice after injection of PBc-UN with or without subsequent NIR illumination. The tumor sites are indicated with arrows. (b) Quantification of the fluorescence intensity at the tumor sites in (a). Data are represented as means ± s.d. (n = 4). *P < 0.05, **P < 0.01.

We then evaluated the NIR light-activated sensing and imaging performance when the nanodevice was delivered by intravenous administration. Animals were given a single i.v. bolus of PBc-UN or Bc-UN in nu/nu mice with 6–8-mm-diameter s.c. tumors. The tumor sites in animals receiving different probes were irradiated or not with NIR light. The PBc-UN + NIR treated group showed stronger fluorescence at tumor site than PBc-UN treated group (Figure 8a). Quantitative analysis showed that the treatment with PBc-UN and irradiation displayed approximately 1.7- and 2.0-fold higher intratumoral signal compared with those without irradiation at 2 h and 4 h, respectively (Figure 8b). The fluorescence intensity of BcUN in the tumor was not enhanced upon irradiation (Figure 8b and Figure S25), suggesting that the signal increase was not because of the irradiation itself, instead, the enhanced fluorescence of PBc-UN in tumors by irradiation was due to photoactivated sensing. Photoactivated imaging was further evaluated by measuring the fluorescence signals in harvested tumors and organs 4 h post-injection (Figure 8c and 8d). The intratumoral fluorescence signal in the PBc-UN + NIR group was much higher than that in nonirradiated animals administered PBc-UC. The mean intratumoral fluorescence in the PBc-UN + NIR group was 2.9- and 3.3fold higher than that in nonirradiated PBc-UN and



irradiated Bc-UN groups, respectively. In the Bc-UN + NIR group, fluorescence at the tumor site was similar to that in the nonirradiated Bc-UN group. There is no significant difference for the fluorescence intensity in the normal organs for all the groups. These data confirmed that remotely activated miRNA imaging in tumor was achieved with this nanodevice.

AUTHOR INFORMATION Corresponding Author *[email protected]

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by NSFC (21822401, 21771044) and the Young Thousand Talented Program.

REFERENCES

Figure 8. NIR light activated miRNA imaging in vivo when DNA nanodevices were delivered by i.v. injection. (a) Wholebody fluorescence imaging of HeLa tumor-bearing mice after intravenous administration of PBc-UN with or without subsequent NIR illumination. Red circles indicate tumor sites. (b) Quantification of the fluorescence intensity at the tumor sites in (a). (c) Ex vivo imaging and (d) region-ofinterest analysis of signal intensities of the tumor and major organs upon different treatments at 4 h post irradiation. Data are represented as means ± s.d. (n = 5). *P < 0.05, ***P < 0.001.

CONLUSIONS In summary, we designed and synthesized a DNA nanodevice that permits NIR light-triggered, spatiotemporally controlled miRNA imaging in live cells and animals. The PBc-UN was constructed through the integration of a UV light responsive beacon probe with UCNPs that acted as the NIR-to-UV transducers to shift the wavelength of activation to NIR light in the biological window. We also verified that the nanodevice can be easily delivered into cancer cells in vitro and in vivo and activated remotely by NIR light for fluorescent detection of miRNAs. We believe that the NIR light-activated miRNAs sensing strategy introduced here will add to the toolbox of techniques for bioanalysis in living systems, which is of significant importance in the diagnosis of human disease, especially cancers.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org. Experimental details and data (PDF)

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A NIR Light-Gated DNA Nanodevice for Spatiotemporally Controlled

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