Photoactivated Nanoflares for mRNA Detection in Single Living Cells

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Photo-Activated Nanoflares for mRNA Detection in Single Living Cells Meihua Lin, Xiaoqing Yi, Fujian Huang, Xin Ma, Xiaolei Zuo, and Fan Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04434 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Analytical Chemistry

Photo-Activated Nanoflares for mRNA Detection in Single Living Cells Meihua Lin,† Xiaoqing Yi,† Fujian Huang,† Xin Ma,† Xiaolei Zuo,‡ and Fan Xia*,†, ‖ †

Engineering Research Center of Nano-Geomaterials of Ministry of Education, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430074, China ‡ Institute of Molecular Medicine, Renji Hospital, School of Medicine and School of Chemistry and Chemical Engineering Shanghai Jiao Tong University, Shanghai 200127, China ‖ Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China ABSTRACT: Gold nanoparticles (AuNPs) have shown great promise as a universal platform for biosensing, and often functionalized with a densely packed DNA for intracellular detection. While DNA-AuNP conjugates, such as nanoflares, have been used for single and multiple mRNA molecules detection in living cells, the target recognition reaction is triggered once they enter into cells, making it impossible to control the initial reaction at the desired time. To solve this problem, we have designed photo-activated (PA) nanoflares for intracellular mRNA analysis with high spatiotemporal control. PA nanoflares consist of a AuNP and photo-responsive DNA hairpin probes. Without UV irradiation, the DNA hairpin could keep unawakened and show no reactivity to target probe. Upon UV activation, the hairpin structures are destroyed and expose the sticky domains, which act as toeholds to mediate strand displacement reactions, making flares release from gold surface and cause an increase of fluorescence. By tuning light irradiation, PA nanoflares for mRNA detection in living cells can be temporally controlled. With the benefit from two-photon laser illumination, PA nanoflares can detect mRNA in selective cells at a desired time point at the single-cell level. Compared to the traditional nanoflares, the novel PA nanoflares have increased the detection sensitivity and achieve intracellular biomarkers detection at the single-cell level with high spatiotemporal control.

Nanoflares appear to be a promising tool for intracellular diagnosis and imaging.1,2 They are composed of a gold nanoparticle (AuNP) functionalized with a layer of densely packed and highly oriented spherical nucleic acids (SNAs), many of which hybridize with short fluorophore-labeled oligonucleotides.3 The labels of fluorophore are in proximity to the gold surface, leading to efficiently quenching of the fluorescence.4 Nanoflares exhibit high levels cellular uptake without the use of transfection agents,5,6 increased DNA stability against enzymatic degradation7 and low cytotoxicity to cells.8 Therefore, nanoflares have been designed to detect intracellular mRNA.3,9-11 Once internalized into cells, nanoflares specifically bind to the target mRNA, forming longer and more stable duplexes. The short reporters, acting as flares, are displaced and liberated from gold surface, resulting in an increase fluorescence signal. The nanoflare architecture provides many advantages for living cell mRNA image, like faster kinetics for target binding than single-stranded SNA-AuNP conjugates,12 lower background fluorescence than molecular beacons, 3 less prone to nonspecific signaling than free DNA.13 However, since cell endocytosis of nanoflares requires hours of incubation and nanoflares are activated by target mRNA once they enter live cells, traditional nanoflares fail to be used in determining the distribution of mRNA in living cells at desired time points, which can provide valuable information about understanding translation, trafficking and degradation of mRNA.10,14-16 To meet the demand for imaging and tracking mRNA with high temporal and spatial resolution, many studies have been

implemented and indicated that the probes could be triggered by a remotely applied physical stimulus in a noninvasive and controllable manner.17-21 Nanomagnetic bead-bound deoxyribozyme has been developed for mRNA sensing in living cells with remote and temporal control by applying external magnetic field.22 Although the probes produce low background fluorescence, the applied magnetic field causes aggregation and free motion of probes, which induces higher local concentration of mRNA and changes the spatial distribution of mRNA. In this regard, light is more attractive and has been successfully used to activate the caged molecular beacons (MBs) for live-cell mRNA imaging with temporal and spatial control.23,24 However, the caged MBs need a carrier to deliver them into cell, which is complicated. Moreover, MB probes are prone to enzymatic degradation within cells, generating a high background signal that is indistinguishable from a true mRNA recognition event. 1,3,25 Therefore, it is desirable to develop an efficient approach for remotely and instantaneously activated sensing of mRNA in living cells with probes having high stability in intracellular environment. Here we report a novel and upgraded nanoflare, consisting of a AuNP and hairpin DNA, which incorporates a photo-cleavable o-nitrobenzyl linker (PC linker) between traditional a thiolated-probe and a flare probe to form a stem-loop structure on the surface of AuNP (Scheme 1), for detection of mRNA in living cells based on the principles of light actuation. The PC linker can efficiently restrict the reaction between target and SNA. However, upon UV irradiation, the PC linker is

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cleaved23,26 and the sticky end of flare, acting as a toehold, is exposed. Then, in the presence of target probes, the toeholdmediated displacement reaction occurs,24 making flares release from the surface of AuNP to cause an increase fluorescence. Moreover, the photo-activated (PA) nanoflare takes advantage of SNAs-AuNP conjugates which are capable of entering livecells without the use of transfection agents, low background fluorescence, high DNA stability against enzymatic degradation, and negligible cytotoxicity.3 The PA nanoflare can freely enter cells and detect mRNA at any desired time by a noninvasive light activation with precise control. What’s more, using twophoton laser,27,28 PA nanoflares can only be activated and lightened up within an interest single cell, but not in adjacent cells, indicating spatiotemporal control of sensing mRNA with single-cell precision. Compared to our previous method of mRNA detection in single living cells,24 this upgraded nanoflare can protect DNA from enzymatic degradation and detect mRNA at the single-cell level without inserting the optical micro/nano fiber or the nanoelectrodes into cells, showing robust and noninvasive detection. EXPERIMENTAL SECTION Chemicals and Reagents. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), HAuCl4•4H2O, cordycepin, lipopolysaccharide (LPS), 6-Mercaptohexanol (MCH), and 3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). 0.2 M Phosphate Buffered Saline (PBS) buffer and 5× TBE were purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China). Foetal bovine serum and Dulbecco's Modified Eagle Medium (DMEM) medium were purchased from Gibco. The human breast cancer cell line MCF-7 was obtained from Cell Bank of the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Other chemicals were obtained from Sinopharm Chemical Reagents, Co., Ltd. (Shanghai, China), and used without further purification. All solutions were prepared with Milli-Q water from a Millipore system. All oligonucleotides were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China), and their sequences are listed as follows: DNA 1: FAM-TCA GTT ACA TTC TCC CAG TTG ATTPC-linker-CTG GGA GAA TGT AAC TGA AAA AAA-SH DNA Target: AAT CAA CTG GGA GAA TGT AAC TGA DNA Target-A5: AAT CAA CTG GGA GAA TGT AAC TGA AAAAA One mismatched Target: ATT CAA CTG GGA GAA TGT AAC TGA Two mismatched Target: ATT CAC CTG GGA GAA TGT AAC TGA Instruments. UV lamp (LUYOR-365, China) was used to activate the PA nanoflares at the power density of 7000 µW/cm2. A Shimadzu UV-2600 spectrophotometer (Shimadzu, Japan) was used to obtain the UV−vis absorption spectra. A FS5 spectrofluorometer (Edinburgh Instruments) was used to measure fluorescence spectra using excitation at 488 nm. Confocal microscopic images were obtained using a Zeiss LSM 880 confocal microscope at 63×magnification. The transmission electron

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microscopic (TEM) images were obtained on a FEI Tecnai G20 U-Twin transmission electron microscope (Hillsboro, OR, USA). MTT assays were performed on a TECAN microplate reader. Photo-Activated DNA Strand Displacement Reaction. The response of DNA hairpin containing PC linker (DNA 1) to UV (λ = 365 nm) irradiation was verified by native polyacrylamide gel electrophoresis (PAGE). DNA 1 mixed with TCEP in 0.01 M PBS was heated at 95 ℃ for 5 min and then cooled to 4 ℃ within 30 s, to form stem-loop structure. DNA 1 was exposed to UV irradiation for 5 min and then incubated with equimolar target DNA for 30 min at room temperature. In order to distinguish the DNA bands, a 5 A segment was added to the DNA Target, named DNA Target-A5. The control experiments were used the same DNA but without UV irradiation. The 20% PAGE was prepared with 1× TBE buffer, and was run at 85 V for 3 h. Preparation of PA nanoflares. The 20 nm AuNPs were first prepared by a classic sodium citrate reduction method.29 Briefly, 100 mL of 0.006 wt% HAuCl4 was heated to boil for 5 min on a hot plate with vigorous stirring. Then, 3 mL of 1 wt% sodium citrate solution was rapidly added under stirring. Boiling was continued for another 30 min until the solution changed into wine red. The reaction was terminated by removing the heating source and cooling to room temperature. The prepared AuNP solution was stored at 4 ℃ for further use. Transmission electron microscopy (TEM) images indicated the particle sizes are 20 ± 1 (nm). The concentration of AuNPs was determined by measuring their absorbance maximum at 524 nm (ε = 9.406 × 108 L mol−1 cm−1). After reduced by TCEP, thiolated DNA 1 was mixed with AuNPs at a ratio 500:1 for 16 h with a shaking at 25 ℃.30 Then 1 M of sodium phosphate buffer (PBS, 1 M of NaCl, 100 mM of Na2HPO4 and NaH2PO4, pH 7.4) was added to the mixture (a total of five salting steps with 2 h intervals between steps) to reach a final concentration of 0.1 M. This salting process was incubated for another 40 h under shaking. After that, the PC nanoflares were prepared, and washed with 0.1 M PBS for 3 times to remove excess DNA through centrifugation. The prepared PC nanoflares were resuspended in 0.1 M PBS, and the concentration of these nanoflares were determined by UV-vis absorption spectra. PC Nanoflares Response to UV Light Irradiation. 0.5 nM PC nanoflares solution with or without 100 nM DNA target was set as a reaction system to response to UV irradiation. Firstly, the kinetic scan mode was selected to monitor the fluorescence changes with time at fixed excitation wavelength of 488 nm, emission wavelength of 520 nm, and 5 s time intervals. After 5 min later, UV lamp (365 nm, 7000 µW/cm2) was used to irradiate the reaction system in situ for 30 s, and the fluorescence recorded the intensity changes for another 5 min without UV irradiation. This alternating fashion was repeated for 10 times. Secondly, the emission scan mode was used to record the fluorescence spectra of different irradiation time at excitation wavelength of 488 nm, emission from 500 nm to 600 nm. The reaction system was exposed by UV lamp for different time and incubated for another 10 min before measurement. The control

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Analytical Chemistry Scheme 1. (A) Illustration of the design and the synthesis of the PA nanoflares, which are gold nanoconjugates functionalized with PC linker incorporated DNA hairpin (DNA 1) oligonucleotide sequences. (B) Representation of general working principle of the photo-activated and specific sequence-responsive PA nanoflares in buffer solution. (C) Represent response of PA nanoflares to target mRNA (MnSOD mRNA as a model) in cells with low or high target mRNA expression level of cells pretreated by cordycepin or LPS respectively, before and after UV irradiation. Scale bar = 20 µm.

samples were conducted as the same process but without UV irradiation. Each experiment was repeated three times. Detection of DNA Target in Solution. 0.5 nM PC nanoflares solution was mixed with different concentrations of DNA target and exposed to 365 nm of UV light (7000 µW/cm 2) for 5 min. After incubation for 1 h, the fluorescence was recorded at room temperature. For specific recognition of PC nanoflares, the same process as the detection of perfect DNA target, but using 100 nM one mismatch or two mismatch target probe. Nuclease Stability. 0.5 nM PC nanoflares solution was incubated with 20 U/mL DNase Ⅰ and monitored by the kinetic scan mode with or without UV irradiation. Cell Culture and Confocal Microscopy Imaging. MCF-7 cells were cultured in DMEM plus 10 % fetal bovine serum, 100 U/mL penicillin-streptomycin and grown in a 100% humidified atmosphere containing 5% CO2 at 37 °C. Cells were plated in a 35 mm2 Petri dish with 10 mm well for 24 h to reach 80% confluence. Then cells were washed three times with PBS buffer and incubated with 2 nM PC nanoflares in culture me-

dium at 37 °C in 5% CO2 for 8 h. After that, cells were washedthree times with PBS to remove the excess probes and immersed in fresh culture medium. For photo regulation, cells were irradiated with UV lamp (365 nm, 7000 µW/cm2) for 5 min and incubated for another 30 min at 37 °C in 5% CO2. Then, confocal imaging was performed on a confocal microscope with a 60× oil-immersion objective. In the experiments for regulation of expression levels of MnSOD mRNA, cells were treated with 150 µg/mL cordycepin or 10 µg/mL lipopolysaccharide (LPS) for 2 h before incubation with PC nanoflares. In the experiments for single cell imaging, after cells incubated with PC nanoflares and washed with PBS, 1 mL PBS was added. An interested single cell was labeled and illuminated by two-photon laser at 740 nm. The cell was imaged at excited laser of 488 nm. RESULTS AND DISCUSSION Principle of Photo-Activated Nanoflares for Sensing mRNA in a Single Live Cell. As illustrated in Scheme 1, the PA nanoflare is constructed by AuNP functionalized with


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photo-responsive hairpin DNA (DNA 1) probes, which are labeled with fluorophores (FAM) and thiols at 5’ and 3’ termini, respectively. The fluorescence of PA nanoflares is quenched due to FAM close to the surface of AuNPs. A DNA 1 probe contains a PC linker between loop and stem, and will expose the sticky end of the flare after light cleavage. The sticky flare sequence is designed for being complementary to the target probe (Figure S1). As a proof-of-concept experiment, manganese superoxide dismutase (MnSOD) mRNA was chosen as a model target, which is related to malignant phenotype and tumor proliferation.31 After entering a cell, PA nanoflares will not react with the target mRNA due to the stable hairpin structure, and remain “unawakened” until the introduction of UV irradiation cleaves PC linkers and exposes sticky domains. Then sticky flares recognize target mRNA and release from gold surface via toehold-mediated strand displacement,32 resulting in fluorescence increased. However, when cells express low level of target mRNA, few PA nanoflares respond to the target and produce negligible intracellular fluorescence after photo activation. Therefore, PA nanoflares can be remotely, temporally and specifically activated the sensing of target mRNA in living cells. Programmable Activation of PA Nanoflares by Irradiation Dosage. Before prepared PA nanoflares, we first validated the cleavage of PC linker in DNA 1 after photo exposure and toehold-mediated strand displacement reaction in the presence

Figure 1. (A) Real-time monitoring of the changes of F/F0 by precise control of UV irradiation state (on) or (off) in the presence or absence of target probes. The red arrows display the mixture was irradiated for 30 s at 365 nm (on), and then left for 5 min without irradiation (off), as the black arrows display the beginning without irradiation. This on and off alternating fashion was repeated for 10 times. (B) Real-time monitoring of the changes of F/F0 in the presence of 100 nM target probe but without UV irradiation. The inset shows the representative fluorescence spectra of PA nanoflares incubated with target probe at the beginning and for 1 h. Representative fluorescence spectra of PA nanoflares respond to different UV irradiation time with target probe (C) and without target probes (D).

of target probe by using native polyacrylamide gel electrophoresis (PAGE). As shown in Figure S1, in the presence of DNA Target but without UV irradiation, there was no new band formed, indicating that hairpin DNA 1 was inactivated and could not hybridize with DNA Target. However, in the presence of DNA Target and with UV irradiation, the band of DNA Tar-

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get was completely disappeared and a new band of short thiolated single-stranded DNA was found, demonstrating PClinker-modified hairpin DNA could be activated by UV light for subsequent toehold-mediated DNA strand displacement reaction. To prepare PA nanoflares, citrate-capped 20 ± 1 nm AuNPs were synthesized (Figure S2) and functionalized with 5’-FAM-labeled and 3’-thiol-labeled photo-responsive hairpin structure DNA 1 through gold-thiol bond. The purified DNAAuNP conjugates show the absorption peak of AuNPs at 525 nm and DNA at 260 nm by UV-vis spectrum (Figure S3), indicated the successful formation of PA nanoflares. The number of DNA 1 assembled on each AuNP was determined to be around 129 by fluorescence (Figure S4). To test the response of PA nanoflares to UV irradiation, the fluorescence intensity was real-time recorded by alternating UV lamp on and off states with precisely controlled irradiation time (Figure 1A). In the absence of target probes, despite of alternating on and off states, the ratio F/F0 (F is the fluorescence intensity at time = t, and F0 is the fluorescence intensity at time = 0) is stable and around 1, demonstrating UV irradiation does not destabilize the PA nanoflares. In the presence of DNA analog of the mRNA target (DNA Target), without UV irradiation, the ratio F/F0 is about 1, and remains constant even though the mixtures were incubated and monitored for 1 h (Figure 1B), suggesting target probes can not bind to PA nanoflares without UV activation. After UV (λ = 365 nm) irradiation for 30 s, F/F0 increases rapidly at first and then gradually increases, indicating UV irradiation can cleave the PC linkers and expose the sticky domains. Target probes can first recognize the sticky domains for DNA branch migration reaction, making sticky flares release from gold surface via toehold-mediated strand displacement, leading to an increase fluorescence. Repeatedly turning UV lamp on for 30 s and off for 5 min for another 9 rounds, F/F0 initially increases rapidly as the increasing alternation times, and then tends to increase slowly. This result indicates the cleavage of PC linker is rapid and efficient, while the toehold-mediated strand displacement on the surface of AuNP is not as fast as in homogeneous solution.12 Then we also evaluated the photo-responsive behavior of PA nanoflares with different independent UV irradiation time by fluorescence spectra. As shown in Figure 1C, in the presence of target probes, the fluorescence increases as UV irradiation time increases from 5 s to 300 s, and saturates at 300 s. However, in the absence of target probes, the UV irradiation causes an ignorable fluorescence enhancement even with 10 min irradiation. These results are in consist with the real-time detection results, indicating PA nanoflares can be programmatically and precisely activated by tuning UV irradiation dosage to signal the presence of the target in a remote manner, and the signal achieves maximum upon irradiation for 5 min at the power density of 7000 µW/cm2. Performance Investigation of PA Nanoflares for Mimic mRNA Detection. After understanding the photoresponsive process of PA nanoflares, we then investigated the sensing performance of PA nanoflares for in vitro target. Upon UV irradiation, the response of PA nanoflares to mimic mRNA (DNA Target) was examined. The results in Figure 2A show the typical fluorescence responses of PA nanoflares to different concentrations of target probes. It is evident that the fluorescence intensity increases with the increasing concentrations of DNA Target from 1 pM to 100 nM and reaches plateau with higher concentrations of target (Figure 2B). The PA nanoflares can detect as low as 1 pM target probe with a linear range from 1 pM to 10 pM. The sensing performance of PA nanoflares is more


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Analytical Chemistry sensitive than traditional sticky nanoflares,10 because the latter was constructed by flare DNA hybridization with singlestranded DNA functionalized AuNPs, and the number of flare probes was limited by the surface hybridization efficiency. 33 The limited number of double-stranded probes also can not make the strands at the surface rigid enough to facilitate target hybridization.34 Therefore, our PA nanoflares can provide more flares and more rigid surface to efficiently increase binding ability of target probes, making PA nanoflares more suitable for sensing lower concentration of mRNAs. Moreover, PA nanoflares have excellent ability to specifically recognize target probes from mismatched ones. As shown in Figure 2C, upon UV activation, PA nanoflares respond with about 5-fold fluorescence enhancement upon target binding and show little increase upon one-base mismatched and two-base mismatched target DNAs.

the viability of cells incubated with PA nanoflares after UV irradiation was as high as that of the control cells (Figure S5), suggesting the negligible cytotoxicity of UV irradiation, PA nanoflares, and UV-activated PA nanoflares. After confirming that the PA nanoflares can work as expected in solution, with high resistance to nuclease cleavage and low toxicity to cells, we investigated the photoactivation and mRNA detection ability of PA nanoflares in MCF-7 cells. cence signal can be observed due to the stability of hairpin DNA. AFigure 3A presents confocal images of cells after incubation with PA nanoflares for 8 h without UV irradiation. No obvious fluoresfter UV irradiation, however, fluorescence emission occurs in the cytoplasm (Figure 3B), suggesting that the photo-activated reaction of the PA nanoflares performed well for mRNA

Figure 3. Confocal images of MCF-7 cells incubated with PA nanoflares under different conditions. Cells were treated with (A, B) culture medium, (C, D) cordycepin, and (E, F) LPS followed by incubation with PA nanoflares and then imaged (A, C, E) without and (B, D, F) with UV irradiation. FAM channels were excited at 488 nm. Scale bar = 20 µm. Figure 2. (A) Representative fluorescence spectra of PA nanoflares responding to various concentrations of DNA Target in vitro after UV irradiation. (B) Corresponding calibration plot of the fluorescence intensity vs the concentration of DNA Target. The inset indicates the linear fitting for different target concentrations from 1 to 10 pM. (C) Representative fluorescence spectra of PA nanoflares responding to 100 nM of various targets, including perfect matched, one base mismatched, and two-base mismatched targets. The group without target is set as the control. All samples have been exposed to UV light. (D) Nuclease stability of PA nanoflares incubated with DNase I with and without UV irradiation. The changes of F/F0 are real-time monitored.

Photo-Activated mRNA Detection in Living Cells. Prior to intracellular mRNA imaging, the ability of AuNPs to protect the hairpin DNA against enzymatic cleavage in cellular environment was tested using DNase I as the model.35 From Figure 2D, we can see that there is no obvious increase in the value of F/F0 when PA nanoflares were incubated with DNase I for about 1 h, irrespective of UV irradiation, indicating that hairpin DNA on the AuNPs can be protected from being digested by DNase I. This nuclease-resistance property is in consistence with previous reports,35,36 stemming from the dense of DNA monolayer on the AuNP surface and low local salt concentration to inhibit enzyme catalyzed hydrolysis.7,37 The stability of PA nanoflares can refrain the false positive signals. We then evaluated the cytotoxicity of PA nanoflares and UV light, using the breast cancer cell line MCF-7 as a model system. Conventional MTT assays were employed in cell viability tests. Results showed that

imaging in living cells. The expression levels of mRNA in cancer cells are different in response to drug therapy.38 It is critical to monitor the change of the expression levels of mRNA to guide therapy. It was reported that cordycepin and lipopolysaccharide (LPS) induced the down regulation and up regulation of MnSOD mRNA expression,23,24,39 respectively. After cordycepin or LPS treatment, PA nanoflares were incubated with MCF-7 cells and confocal imaging was performed. Without UV irradiation, the hairpin DNA on the gold surface keeps unawakened state, and negligible fluorescence signal was observed in any sample (Figure 3C and 3E). In contrast, UV irradiation can activate the ability of PA nanoflares to detect target mRNA. As shown in Figure 3D and 3F, cells treated with cordycepin show lower fluorescence while cells treated with LPS show higher fluorescence, both compared with the untreated cells. Figure S6 shows the relative fluorescence intensity of FAM under different conditions. These results are in consistence with previous reports,23,24,39 and indicate the fluorescence intensity correlates with the expression level of the target mRNA in living cells. Therefore, PA nanoflares have the capacity for measuring changes of gene expression levels in cancer cells with precise temporal control. Compared to the common method of mRNA detection by fluorescence in situ hybridization (FISH), which needs fixation and pretreatment of cells, 40 PA nanoflares can be used for visualization of mRNA in living cells at a desired time point of the cell life-cycle. Although cell-to-cell variations of mRNA affected the biological processes has been demonstrated.16,41,42 It is a major challenge to detect mRNA with spatiotemporal control at the single-


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cell level. With the benefit from a femtosecond two-photon laser, which can provide improved three-dimensional spatial localization and increased imaging depth,43 the activation of PA nanoflares could be temporally and spatially controlled, so we can selectively activate the designated cells at the single-cell level. PA nanoflares have advantages of high levels cellular uptake, DNA stability against nuclease, and activation by spatial and temporal control. We challenged PA nanoflares for sensing single-cell mRNA by extracting single cells of interest via a two-photon laser illumination.44,45 As shown in Figure 4A, before two-photon irradiation, the designated cell was labeled for laser illumination and excited the FAM fluorescence, but there was no detectable signal in all cells (Figure 4A). However, after

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PA nanoflare for mimic mRNA detection is more sensitive than traditional sticky-flare, due to the large number of flares making the strands on gold surface much more rigid for enhanced target binding. Third, PA nanoflares for mRNA detection in living cells are easily controlled by photocleavage with high temporal resolution, and PA nanoflares can also sense changes in mRNA expression levels in cancer cells. By means of two-photon laser, the spatial activation of PA nanoflares can detect mRNA in selective cells at the single-cell level with temporal control. Moreover, PA nanoflares have great potential to detect other targets and multiple targets by rational design DNA sequence.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supplementary experimental procedures of quantification of DNA 1 loaded on AuNPs and cell viability assay, supplementary figures (Figure S1- S8) (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: +86-27-67885201. Fax: +8627-67885201.

ORCID

Figure 4. Confocal images of MCF-7 cells incubated with PA nanoflares before (A, C) and after (B, D) selectively activated by two-photon laser at 740 nm. Only single cell was selected in (A, B), while four random cells were selected in (C, D) for two-photon illumination at the desired time point. FAM channels were excited at 488 nm. The fluorescence was observed within the red circle after two-photo laser selective irradiation. The area surrounded by the red line circle presents the irradiation area by two-photo laser. Scale bar = 20 µm.

two-photon irradiation, the fluorescence was only found within the illuminated cells (Figure 4B) and the fluorescence intensity increased at the single-cell level (Figure S7), but not in adjacent cells, suggesting that PA nanoflares can spatiotemporally detect mRNA with single-cell precision. Also, our result is in consistence with previous reports that AuNP-DNA conjugates could be used for intracellular mRNA detection.3,5,9-11,35 Conventional nanoflares as control probes shown that two-photo laser illumination would not cause the probes aggregated in a single living cell (Figure S8). Due to the precise spatial localization of twophoto laser, PA nanoflares can also sense many separated cells at the same time at the desired time point by illumination only these selected cells (Figure 4C and 4D) with fluorescence increased (Figure S7), but not in surrounding cells. These results demonstrate that the PA nanoflare is powerful for mRNA analysis in single living cells with high spatiotemporal control. CONCLUSION In conclusion, we have demonstrated a novel photo-activated nanoflare for intracellular mRNA detection. The PA nanoflares not only have advantages of traditional nanoflares, such as efficient fluorescence quenching, cell internalization and cellular stability, but also show their new advantages. First, PA nanoflares can be programmatically and precisely activated by controlling light irradiation dosage in a remote manner. Second,

Meihua Lin: 0000-0001-7616-7358 Fujian Huang: 0000-0002-7777-1589 Xiaolei Zuo: 0000-0001-7505-2727 Fan Xia: 0000-0001-7705-4638

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by the Fundamental Research Funds for the Central Universities, China University of Geosciences (Wuhan) (CUG170660), the Natural Science Foundation of Hubei Province of China (2018CFB169), the National Basic Research Program of China (973 Program, 2015CB932600), the National Key R&D Program of China (2017YFA0208000, 2016YFF0100800), the National Natural Science Foundation of China (21525523, 21722507, 21574048), and the Fok Ying-Tong Education Foundation, China (151011).

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