DNA-Conjugated Amphiphilic Aggregation-Induced Emission Probe

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DNA-conjugated Amphiphilic Aggregation-induced Emission Probe for Cancer Tissue Imaging and Prognosis Analysis Xudong Wang, Jun Dai, Xuehong Min, Zhihua Yu, Yong Cheng, Kaixun Huang, Juliang Yang, Xiaoqing Yi, Xiaoding Lou, and Fan Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01456 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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Analytical Chemistry

DNA-conjugated Amphiphilic Aggregation-induced Emission Probe for Cancer Tissue Imaging and Prognosis Analysis Xudong Wang,a# Jun Dai,a# Xuehong Min,a Zhihua Yu,a Yong Cheng,a Kaixun Huang,a Juliang Yang,b Xiaoqing Yi,b Xiaoding Lou*b and Fan Xia*ab a

Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Department of Obstetrics and Gynecology, Tongji Hospital Tongji Medical College, Institute of Pathology of Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430074, P. R. China. b Engineering Research Center of Nano-Geomaterials of Ministry of Education, Faculty of Materials Science and Chemistry, China University of Geosciences, 388 Lumo Road, Wuhan 430074, P. R. China Fax: +86 027 67885201. E-mail: [email protected]; [email protected]; [email protected] ABSTRACT: Detection of ultralow concentration of mRNA is important in prognosis of gene related diseases. In this study, a DNA-conjugated amphiphilic aggregation-induced emission probe (TPE-R-DNA) was synthesized for cancer tissue imaging and prognosis analysis based on exonuclease III-aided target recycling technique. TPE-R-DNA comprise two components: a hydrophobic component that serves as the “turn-on” long wavelength fluorescence imaging agent (TPE-R-N3); a hydrophilic single DNA strand (Alk-DNA) which acts as specific recognition part for target mRNA. In the absence of target mRNA, TPE-R-DNA was almost no fluorescence due to the highly water solubility. Conversely, the TPE-R-DNA was digested by exonuclease III (Exo III) in the presence of MnSOD mRNA to release the hydrophobic fluorogens (TPE-R-AT). Subsequently, TPE-R-AT formed aggregates, and therefore, fluorescence signal was distinctly observed. For the first time, the structure of the hydrolysis product (TPE-R-AT), containing two bases A and T, was proved by mass spectrum (MS) and high performance liquid chromatography (HPLC). Moreover, the detection limit towards mRNA could be achieved in as low as 0.6 pM. Furthermore, the fluorescent signal can be used to confirm the MnSOD mRNA expression level in cancer tissue. The MnSOD mRNA expression in renal cancer was lower than in renal cancer adjacent tissue. In particular, the expression level was analyzed to predict prognosis of cancer patients. Our results demonstrate that a shorter survival time was evident among patients in lower MnSOD mRNA expression. Thereby, it indicates great potential for the development of ultrasensitive biosensing platform for the application in disease prognosis.

Prognosis in cancer is essential for treatment decision making, patient counseling, and inclusion in clinical trials. Most commonly, prognosis has been found to be based on the three main prognostic determinants used in routine practice, i.e., tumor size and differentiation, lymph node involvement, and metastasis.1, 2 The above staging system can provide useful information for prognosis as well as guide therapy.3 However, current cancer staging systems have been proven to be less precise outcome prediction in an individual patient; in addition, new information including prognostic markers or more complex biologic information cannot easily be incorporated into staging systems.4 In view of this, a potential prognostic marker is urgently needed for the prognosis of cancer. Over the past few years, researchers have demonstrated that intracellular cancer-related mRNA is a specific marker of cancer and its relative expression level can yield valuable information for the disease progression and prognosis. Thus, a number of approaches have been successfully devoted to analyzing endogenous mRNA, such as in situ hybridization (ISH),5-7 polymerase chain reaction (PCR)8 and northern blot (NB) analysis.9 Typically, Lewis’s group developed an in situ hybridization technology to characterize mRNA expression profile in oropharyngeal squamous cell carcinomas (OSCCs) for patient outcomes.10 Shi et.al reported a mRNA gene ex-

pression signature using quantitative real-time polymerase chain reaction (qRT-PCR) method for the prediction prognosis in patients.11 Petersen and coworkers designed a northern blot (NB) assay to specially recognize the PDCD4 mRNA in human lung cancer.12 However, these techniques still subjected to different drawbacks including large sample consumption, time-consuming or insufficient sensitivity. Fluorescence methods have attracted tremendous interest because of their advantages in term of appropriate sensitivity, high selectivity, rapid, simplicity and in situ response.13-25 Among them, several technologies have been incorporated for mRNA analysis including molecular beacons16, 26-29 and nanoflares.30-33 Normally, the combination of fluorophore and quencher group in nucleic acid probes is a commonly utilized strategy for biological detection. However, the coexistence of both fluorophore and the quencher group, not only induce the difficulties to design the structure, but also bring the inconvenience of organic synthesis. In view of this, it would be strong advocated if there is a group of fluorogens can be utilized to predigest the design and synthesis process via their intrinsic optical property. Aggregation-induced emission fluorogen (AIEgen) is a new class of organic dyes, which display almost nonemissive in solutions but show bright fluorescence in aggregate state.34, 35

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AIEgen has proposed a solution for the construction of novel biosensor.36-43 Owing to aggregation-induced emission characteristic of fluorophore, the probe without quencher could reduce synthetic procedures and simplified the design proposal. In our prior study, we have developed a series of fluorescent probes without quencher, which can be successfully applied to detect telomerase, miRNA and protein in living cells.44-48 However, these fluorescent probes with the emissions in the lower wavelength region of the visible spectrum are subjected to the interference of biomolecule especially in tissues as the self-absorption and auto-fluorescence are high. To monitor the analysis in tissues, probes with emission in the red region, which have less background auto-fluorescence, are necessary to be designed.49 In this work, we proposed a DNA-conjugated amphiphilic aggregation-induced emission probe (TPE-RDNA) with long wavelength red emissive AIEgen without quencher for in situ monitoring of mRNA expression in living cells and cancer tissues. Moreover, the AIEgen in this probe emit red fluorescence, instead of these fluorescent probes with the emissions in the lower wavelength region of the visible spectrum, which are subjected to the interference of biomolecule especially in tissues due to the high self-absorption and auto-fluorescence. Thus, with above distinguishing features, TPE-R-DNA can be applied to predict prognosis of cancer patients via detecting the expression level of mRNA in tissue chips. To the best of our knowledge, the detection of mRNA based on the Exo III-aided target recycling process in cancer tissues for prognosis assessment have not been explored until now. As a consequence, this method provided an ultrasensitive biosensing platform for mRNA analysis and showed high potential for the prediction of the overall survival of patients with gene-related diseases.

EXPERIMENTAL SECTION Reagents. Dimethyl sulfoxide (DMSO), acetonitrile, copper (I) bromide and sodium ascorbate were obtained from Aladdin Industrial Corporation (Shanghai, China) and used as received without any further purification. Water used throughout all experiments was obtained from a Millipore filtration system. Other chemicals were ordered from Sigma-Aldrich and used as received without any further purification. The nuclease exonuclease III, NEB buffer, RNase inhibitor, thrombin, Bst DNA polymerase, S1 nuclease, and DEPC-treated water was bought from TaKaRa Bio Inc. (Dalian, China; DEPC = diethylpyrocarbonate). The exonuclease III specifically catalyzes the stepwise removal of mononucleotides from 3'-hydroxyl termini of duplex DNA. RNase inhibitor and RNase form 1:1 complex, and then inhibit RNase activity. S1 nuclease is a single-strand-specific endonuclease that hydrolyzes singlestranded RNA or DNA into mononucleotides. All oligonucleotides were purchased from Sangon Biotech (Shanghai, China) (Table S1). Instruments. 1H spectra were measured on a Bruker ARX 400 NMR spectrometer using chlroform-d (CDCl3-d) as solvent and tetramethylsilane (TMS) as internal reference. Highresolution mass spectra (HRMS) were obtained by using a Bruker microTOF II mass spectrometer system operating in a MALDI-TOF mode. High performance liquid chromatography (HPLC) was performed by using Wufeng 100 for semipreparative HPLC under the test wavelength of 254 nm. Ultraviolet spectra were conducted on an Agilent Cary 60 UV-visible spectrometer. The fluorescence spectra were recorded using an Agilent Cary Eclipse fluorescence spectrophotometer. Confo-

cal laser scanning microscopy images were recorded using an Olympus Fluoview FV1200 confocal laser scanning microscope or a Zeiss LSM 880 confocal laser scanning microscope. Partical size of the liberated TPE-R were determined by dynamic light scattering (DLS) using a Malvern Instruments Zetasizer Nano ZS90. MTT assay was obtained on an Infinite M200 PRO Microplate Reader (Tecan Austria). Electrophoresis Experiment. PAGE (10%, w/w) in the buffer of 1 × TBE (pH 7.9, 9 mM Tris-HCl, 9 mM boric acid, 0.2 mM EDTA) was used to test different samples at the voltage of 100 V at 25 °C. After separation, the PAGE gels were photographed using gel image system. Synthesis of TPE-R-N3. For further details, please see the Supporting Information. Synthesis of TPE-R-DNA Conjugates via Click Reaction. The preparation of TPE-R-DNA conjugates is described in the Scheme 1. In a typical synthesis, a solution of sodium ascorbate (625 nmol) in 50 µL water under argon was mixed with cuprous bromide (1250 nmol) in 100 µL DMSO. Afterwards, the alkyne oligonucleotide (Alk-DNA, 27.2 nmol) in 100 µL water was introduced into the above solution followed by the TPE-R- N3 (272 nmol) in 50 µL DMSO. The reaction mixture was kept stirring under argon at room temperature for 24 h. The crude reaction mixture was then purified on reversedphase HPLC to give 30% isolated yield. Cell Culture. HeLa and MCF-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37℃ in a humidified atmosphere containing 5% CO2, respectively. Cell density was determined using a hemocytometer before experimentation. Cytotoxicity assay. The cytotoxicity of TPE-R-DNA on MCF-7 cells were assessed using standard MTT assays. First, 5×103 cells/mL MCF-7 cells were seeded onto 96-well plates and grown for 24h. The cells were treated with various concentrations of TPE-R-DNA (0-10 µM) for 24h. After that, a total 20 µL of MTT solution (5 mg/mL) was added into every well incubated at 37 ℃ for 4h. Finally, the supernatants were aspirated and then 150 µL of DMSO was added to each well. The absorbance values of the wells at 570 nm was performed on a microplate reader. Incubation living cells with probe. HeLa and MCF 7 cells were cultivated on 35-mm confocal dish, at an initial density of 4 × 104 cells/dish, and cultivated for 24 h prior to experiment. PBS (0.1 M, pH 7.4) was used to wash cells for three times. Subsequently, the cell culture medium was changed to fresh Opti-MEM for 2h. The lipofectamine 2000 Reagent (10 µL) or the TPE-R-DNA (6.6 µM) and exonuclease III (1.33 U/µL) were separately diluted in 140 µL of Opti-MEM at 37 °C for 5 min. Subsequently, the above solutions were mixed and let stand for 20min. The above mixture was added in the dishes. After reaction at 37 ℃ for 1h, washing buffer was employed to wash the cells 3 times. After washing with PBS three times at room temperature, and then, they were added to 1 mL of PBS culture medium before imaging. Confocal Fluorescence Imaging. Fluorescence imaging of live cells was performed on an Olympus Fluoview FV1200 confocal laser scanning microscope or a Zeiss LSM 880 confocal laser scanning microscope with a 60×oil-immersion objective. The fluorescence images were collected at blue channel (Hoechst) ranging from 400 to 500 nm with an excitation


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Analytical Chemistry at 405 nm, and the red channel (TPE-R-DNA) ranging from 550 to 650 nm with an excitation at 488 nm. Quantitative real-time PCR analysis. Quantitative realtime PCR analysis was obtained by using the Power SYBR Green PCR Master kit (Applied Biosystem). The sequences of PCR primers are shown in Supplementary Table S1. Amplification was carried out on Stepone Plus real time PCR system. Clinical Sample Analysis. The tissues were incubated with the above detection mixture for 1h at 37 ℃. After incubation, the tissues were washed with washing buffer for 3 times. The tissues were visualized under an Olympus FV1200 confocal scanning system with an objective lens (60×). Histological Analyses. Specimens were fixed in 10% neutral-buffered formalin, dehydrated in graded alcohols, and then embedded in paraffin. Sections (1 mm) were cut and then stained with hematoxylin and eosin (H&E).

Scheme 1. (a) Synthetic route of TPE-R-DNA and (b) schematic representation of TPE-R-DNA probe for detection of MnSOD mRNA.

RESULTS AND DISCUSSION Synthesis and design principle of TPE-R-DNA. The synthetic routes toward TPE-R-N3 were illustrated in Scheme S1. Firstly, azide-functionalized pyridine (1) and the aldehydefunctionalized TPE-R (2) were prepared according to the procedures in literatures.48, 50 Then, a mixture of 1 (103.5 mg, 0.5 mmol) and 2 (210.1 mg, 0.5 mmol) in dry EtOH (15 mL) was refluxed under nitrogen for 48 h to obtain TPE-R-N3 (3) through vinyl functionality. Finally, a copper-catalyzed azidealkyne click reaction between TPE-R-N3 and Alk-DNA took place in the presence of in a DMSO/water (v/v=1/1) mixture containing CuBr/sodium ascorbate, affording TPE-R-DNA at room temperature as depicted in Scheme 1a. TPE-R-DNA is composed of two parts: a hydrophobic fluorogen (TPE-R-N3) with aggregation-induced emission characteristic and a hydrophilic single strand DNA (Alk-DNA). The TPE-R-N3 with red fluorescence served as the signal molecule due to its higher penetrability to biological samples compared with the shorter wavelength. The Alk-DNA was designed as the recognition portion, including the partial complementary base sequence of the MnSOD mRNA. Manganese superoxide dismutase (MnSOD) catalyzes the conversion superoxide radicals to hydrogen peroxide and oxygen that can protect cells from the damage arisen from reactive oxygen species. In this process, the significant event is that MnSOD mRNA is transcribed into MnSOD. The MnSOD mRNA expression level is regarded as a significant parameter for cancer diagnosis as well as prognosis. Therefore, TPE-R-DNA involves both target binding and fluorescent signal response property. TPE-RDNA showed weakly emissive as a result of the attachment of hydrophilic single strand DNA to yield the molecularly dissolved state. In the presence of target, the TPE-R-DNA would bind to target, leading to duplex conformation. Exo III is a kind of exonuclease that can catalyze the stepwise hydrolysis of mononucleotides of double-stranded DNA in the 3′ to 5′direction, and limited activity on single-stranded DNA or 3′ protruding termini of double-stranded DNA. As a result, TPER-DNA incorporated in the duplex was completely digested using Exo III in the direction from 3′-ends to 5′-ends. The digestion process results in the releases of the target and generates the hydrophobic fluorogens. Since they exhibit poor dissolvability in aqueous medium, the emission could be switched on when aggregates. In the meantime, the released target could repeatedly hybridize with another TPE-R-DNA

for subsequent the recycling hydrolysis reaction. Thus, the designed fluorescence strategy based on based on exonuclease III-aided target recycling technique can be achieved for highly sensitive quantification of the expression of mRNA in tissue. Characterization of the TPE-R-DNA. The intermediate products and TPE-R-DNA were characterized by high performance liquid chromatography (HPLC), high resolution mass spectra (HRMS) and ultraviolet visible spectroscopy (UV-Vis) to confirm its chemical structure and purity (Figure 1 A-C and Figure S1). TPE-R-DNA was purified by HPLC, and the appearance time of TPE-R-DNA was earlier than TPE-R-N3 (Figure 1A). As outlined in Figure 1B, the mass spectra manifested a peak at m/z 5705.5, which confirmed the successful synthesis of TPE-R-DNA. The Uv-vis absorption spectra of TPE-R-N3 and TPE- R-DNA were carried out. As can be seen in Figure 1C, the absorption peak of TPE-R- N3 located at around 328 nm and 428 nm. The TPE-R-DNA exhibited three peaks located at around 260nm, 338nm and 428nm. Besides the characteristic absorption of TPE-R- N3, an obvious peak centered at 260 nm was also observed, which was ascribed to the characteristic absorption spectra of DNA. The absorption spectra proved the success of the coupling between TPE-R-N3 and Alk-DNA. Thus, these results verified the TPE-R-DNA was successfully synthesized. Feasibility Analysis. To assess the stability of TPE-RDNA, two experiments in biological environments were carried out. As illustrated in Figure S2A, the fluorescence of TPE-R-DNA change slightly in salt, enzyme, pH, ATP, cell lysate and serum for 1h, demonstrating that the probe was not obviously degrade. Figure S 2B depicts that only one band from gel electrophoresis was observed. It was evident that the TPE-R-DNA stability was sufficient for application in biological environment. In order to investigate the validity of the proposed



Figure 2 (A) Fluorescence emission spectra of TPE-R-DNA (10 µM) in the presence of Exo III (300 U) and different concentration of DNA target (0-1000 pM), (B) BSA (10 mg/L), trypsin (10 nM) and Bst DNA polymerase (Bst) (16U) were used instead of the Exo III under the same experimental conditions. (C) Specificity tests demonstrate that the assay could distinguish the perfect match (PM) targets with single/triple mismatched targets. (D) The cytotoxicity of TPE-R-DNA was tested with MCF-7 cells by MTT assay. MCF-7 cells were incubated with TPE-R-DNA of different concentration (0, 0.1, 0.5, 1, 3, 6, 8, 10 µM) for 12h.

Figure 1 (A) High performance liquid chromatography (HPLC) results of TPE-R-DNA and TPE-R-N3. (B) Mass spectra of TPER-DNA. (C) Uv-vis spectra of TPE-R-N3 (red) and TPE-R-DNA (black). (D) High performance liquid chromatography (HPLC) results of TPE-R-AT (red) and mass spectra of TPE-R-AT (black) (E) Chemical structure of TPE-R-AT (F) Dynamic light scattering measurements of TPE-R-DNA (red) and TPE-R-DNA incubated with mRNA and Exo III (black). (G) The fluorescence intensity of the TPE-R-DNA in the presence of S1 nuclease (10 U) is 516% higher than that in the absence of it.

method, the gel electrophoresis has been used for analyzing the degradation of TPE-R-DNA probe by Exo III (Figure S4). Lane e showed the hybridization of target and TPE-R-DNA, illustrating that the duplex cannot be consumed by Exo III. In contrast, only one band of the mixture of mRNA and TPE-RDNA was observed in the presence of Exo III, suggesting that TPE-R-DNA probe can be cleaved by Exo III (Figure S4, lane f). Furthermore, Figure 1D shows the mass spectrum of the hydrolysis product of TPE-R-DNA manifested at 1437.5416 which was TPE-R-AT. The appearance time of hydrolysis product (Figure 1D) is 16.73 min which is between TPE-R-N3 and TPE-R-DNA, further proved that the hydrolysis product is TPE-R-AT. In addition, dynamic light scattering analysis demonstrated that average hydrodynamic diameter of TPE-R-

DNA was about 40 nm (Figure 1F, black), however, TPE-RAT aggregates was around 340 nm (Figure 1F, red). Moreover, upon addition of S1 nuclease (10 U), the fluorescence intensity significantly increased because of the formation of hydrophobic TPE-R-AT aggregates (Figure 1G). Therefore, the aforementioned results clearly confirmed that the feasibility of the TPE-R-DNA probe can be used for simultaneous detection of mRNA. In vitro response of the TPE-R-DNA. To demonstrate the performance of our method, various concentration of synthetic DNA target was added to the reaction system. As shown in Figure 2A, the fluorescent intensity was observed as a function of concentrations of DNA target. In the absence of DNA target, the TPE-R-DNA probe exhibited a slight fluorescence emission, thus indicating the good solubility of TPE-R-DNA probe in aqueous medium. Upon the addition of DNA target, the fluorescence intensity at 620 nm enhanced progressively with the concentration of mRNA from 0 to 1000 pM. A maximum 4.5-fold increase in fluorescence intensity could be obtained with the concentration of 1000 pM (Figure 2A, inset). In the meantime, the fluorescence intensity increased linearly with the concentration of DNA target over the ranges from 1 to 1000 pM, and the detection limit was estimated to be 0.6 pM (R2=0.95), which was comparable to or lower than the previously reported methods (Table S2).51-54 The high sensitivity should be owe to the signal amplification. Next, we also explored the importance of Exo III to our proposed strategy. As illustrated in Figure 2B and Figure S4, there was significant difference of fluorescence signal with the aid of Exo III. On the contrary, in the presence of three kinds of proteins including BSA, trypsin and Bst DNA polymerase. The fluores-


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Analytical Chemistry cence signal was negligible, indicating that TPE-R-DNA only can be

Figure 3 CLSM images of mRNA in Hela cells under different conditions. Cells were pretreated with (A) PBS, (B) 150 µg/mL cordycepin or (C) 10 µg/mL LPS for 2 h and then incubated with the TPE-R-DNA (6.6 µM) and Exo III (1.33 U/µL) for 1h under the standard cell culture conditions, respectively. The relative fluorescence intensity of (D) PBS, (E) cordycepin or (F) LPS. The fluorescence images were collected at blue channel ranging from 400 to 500 nm with an excitation at 405 nm, and the red ranging from 550 to 650 nm with an excitation at 488nm. Scale bar: 20µm.

digested by Exo III. The above experiments demonstrated that Exo III played a critical role for our assay. Finally, the specificity of the strategy was evaluated. The TPE-R-DNA probe was verified by treating TPE-R-DNA with DNA strands containing the perfect match, single-base mismatched strand (SM), and three-base mismatched strand (TM) under the same concentration (10 µM). As indicated in Figure 2C and Figure S5, the perfectly matched target can be well discriminated from both SM, and three-TM. The perfectly matched strand showed 2.4 fold and 3.6-fold higher signal than single-base mismatch strand and three base mismatch sequence, respectively, arising from the limited digestion property of Exo III towards the mismatched duplex. These results demonstrated that the TPE-R-DNA probe could effectively recognize target. Live cell mRNA imaging using the TPE-R-DNA. In view of the favourable sensitivity and selectivity towards target in vitro system, the capability of TPE-R-DNA to visualize mRNA inside living cells using confocal laser fluorescence microscopy was examined. First, cytotoxicity of TPE-R-DNA (Figure 2D) and Exo III (Figure S6) was conducted on MCF-7 Cells by MTT assay. No obvious cytotoxicity was observed as the viability of MCF-7 cells even in the concentration of TPER-DNA up to 10 µM. The viability of MCF-7 cells was over 85% even in the presence of 2 U/µL Exo III, confirming the low cytotoxicity of Exo III. Additionally, Figure S7 showed that Exo III could enter into cell successfully. It was well known that cordycepin and lipopolysaccharide (LPS) could induce downregulation and upregulation of MnSOD mRNA,

respectively. Thus, we utilized cordycepin and LPS for MnSOD mRNA disturbance experiments. Figure 3A and D shows confocal laser fluorescence images of Hela cells after treatment with only the TPE-R-DNA and Exo III at 37 °C for 1 h. However, when the cells were pretreated with cordycepin

Figure 4 CLSM images of mRNA in MCF-7 cells under different conditions. Cells were treated with (A) PBS, (B) 150 µg/mL cordycepin or (C) 10 µg/mL LPS for 2 h and then incubated with the TPE-R-DNA (6.6 µM) and Exo III (1.33 U/µL) for 1h under the standard cell culture conditions, respectively. The relative fluorescence intensity of (D) PBS, (E) cordycepin or (F) LPS. The fluorescence images were collected at blue channel ranging from 400 to 500 nm with an excitation at 405 nm, and the red ranging from 550 to 650 nm with an excitation at 488nm. Scale bar: 20µm

for 2h, the decreased fluorescence signal could be visualized (Figure 3 B and E). Moreover, the cells were treated with cordycepin for 1h, 2h and 3h. The fluorescence continuously decreased over time (Figure S8). By contrast, the fluorescence signal increased after pretreatment with LPS compared with the untreated cells (Figure 3C and F). Additionally, the cells were preincubated with LPS for 1h, 2h and 4h. As time went on, the fluorescence gradually increased (Figure S9). To further verify its universality, TPE-R-DNA was incubated with MCF-7 cells. As displayed in Figure 4A and D, the probe exhibited the brightest red fluorescence signal in cells with LPS treatment, suggesting its highest intracellular MnSOD mRNA expressional level compared to those with PBS or cordycepin treatment. In control experiments, the cells were treated with cordycepin at different time points under the same conditions. The fluorescence decreased with the increasing treatment time (Figure S10). In the meanwhile, the cells exhibited the significant fluorescence increased with the incubation time of LPS (Figure S11). The imaging results were further validated by CLSM with 10×objective for mRNA levels (Figure S12 and Figure S13). As can be seen in Figure S14, the fluorescence image with 5×objective consistent with the above confocal image results. The results obtained from PCR further suggested that

cordycepin can decrease MnSOD mRNA levels and LPS can increase MnSOD mRNA levels (Figure S15). These results showed that the intracellular fluorescence signal is indeed, aroused by mRNA and TPE-R-DNA could undoubtedly monitor the intracellular mRNA level.



mRNA imaging in tissue using TPE-R-DNA. Before the practical application of TPE-R-DNA probe, the photostability were investigated. As shown in Figure 5A and Figure 5B, signal remaining of TPE-R-DNA in liver cancer tissue was up to 95% even after 48 scans, which exhibited excellent photostability as well as high resistance against photobleaching. We further measured the expressions of MnSOD mRNA in tissues

CLSM. As outlined in Figure 5 B-E, different tissues incubated with TPE-R-DNA probe also displayed strong fluorescence. The results were also consistent with the results of the representative photomicrographs of hematoxylin and eosin (HE). The results demonstrated that the practicability of the probe for the detection of mRNA in tissues. We gained further insight on the expression level of mRNA in renal cancer, we examined MnSOD mRNA expression on clinical tissue specimens from 90 patients (both cancer tissue samples and adjacent normal tissue samples). Figure 6 A and Figure S16 showed the confocal images of renal cancer and its

Figure 6 CLSM images (A) of Renal cancer (ca.) and its adjacent tissue (AT). Fluorescence intensity (B) of Renal cancer adjacent tissue. (C) Kaplan–Meier survival curves of patients classified into high- and low-expression groups based on the MnSOD mRNA signature. The fluorescence images were collected from 550 to 650 nm with an excitation at 488nm. Scale bar: 1 mm. Figure 5 (A) Time course of CLSM images of liver cancer tissue. (B) Signal remaining (%) of fluorescence intensity of TPE-RDNA in liver cancer tissue with increasing number of scans. CLSM images of renal cancer (C), lung cancer (D), intestinal cancer (E), gastric cancer (F) and liver cancer (G) and representative HE staining images of the corresponding tumor tissues. The fluorescence images were collected at blue channel ranging from 400 to 500 nm with an excitation at 405 nm, and the red ranging from 550 to 650 nm with an excitation at 488nm. Scale bar: 20µm.

using our proposed strategy. As demonstrated in Figure 5C, confocal laser scanning microscopy results showed that the renal cancer tissue incubated with the TPE-R-DNA probe dis played the distinct red fluorescence signal. Additionally, we evaluated this assay to detect mRNA in other tumor tissues to confirm our method have universality in mRNA detection. Next, we tested the probe treated with four kinds of tumor tissues including lung cancer, intestinal cancer, gastric cancer and liver cancer. These four types of tissues were incubated with TPE-R-DNA probe for 1h and then imaged by the

adjacent normal tissue. The mean fluorescence intensity of adjacent normal tissue is 1.46 times higher than the fluorescence intensity of renal cancer tissue (Figure 6B), suggesting that there is higher mRNA expression in adjacent normal tissues than in tumor tissues. The results were consistent with the previous literature.55 To further evaluate whether expression level of MnSOD mRNA can predict prognosis, we next performed a survival analysis on 90 patients. In this experiment, patients were divided into two equal groups (low and high) by using the median values of MnSOD mRNA in Table S1 as cutoff. The overall survival was analyzed with the KaplanMeier method. Analysis of MnSOD mRNA expression in time to overall survival of the 90 patients with renal cancer are outlined in Figure 6C. It was worth noting that low MnSOD mRNA expression of patients suffering from renal cancer appear to be related with a decreased survival rate compared to those with high level of MnSOD mRNA (P

DNA-Conjugated Amphiphilic Aggregation-Induced Emission Probe

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