Continuous Monitoring of Specific mRNA Expression Responses with

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Continuous monitoring of specific mRNA expression responses with a FRET-based DNA nano-tweezer technique that does not require gene recombination Hajime Shigeto, Keisuke Nakatsuka, Takeshi Ikeda, Ryuichi Hirota, Akio Kuroda, and Hisakage Funabashi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02710 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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Continuous monitoring of specific mRNA expression responses with a FRET-based DNA nano-tweezer technique that does not require gene recombination. Hajime Shigeto†, ‡, Keisuke Nakatsuka‡, Takeshi Ikeda‡, Ryuichi Hirota‡, Akio Kuroda‡, and Hisakage Funabashi*,† †Institute for Sustainable Sciences and Development, Hiroshima University, Higashihiroshima, Hiroshima 739-8511, Japan ‡Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashihiroshima, Hiroshima 739-8530, Japan. ABSTRACT: This report discusses the feasibility of continTareget mRNA Closed state uously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resoOpen state FRET signal nance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open FRET-based Excitation Fluorescence Cy3 Cy5 DNA-NT state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modLive-cell imaging ified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 Living cell and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semi-quantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner.

Introduction Single cell analysis of gene expressions reveals that behaviors of individual cells are different even when they are in the same cellular mass, indicating approaches to understanding biological phenomenon as a group1-4. Recent advances in molecular biological research methods such as polymerase chain reactions (PCRs) enable the single cell analysis of specific gene expression status by killing the cells and extracting their components. However, with such methods, it is impossible to analyze subsequent cellular responses, which are important because cells often behave in different ways. Photo-imaging analysis is a powerful approach to the non-destructive analysis of cellular response. Conventionally, reporter proteins such as luciferase expressed under control of a promoter of interest allow us the investigation of the promoter activity with luminescence in a non-destructive manner5-8. Also a split fluorescent-protein complementation system has widely been used to monitor mRNA dynamics by tracing the fluorescence in a non-destructive manner9-12. However, these techniques require gene recombination, and thus it is difficult to apply in certain cases such as a biopsy and cellular regenerative medicine. It is usually very difficult to culture the primary cells collected from the patient13,14, and the integration of such extracellular

genes should be avoided15, respectively. The molecular beacon (MB)16 is one of the strongest candidates to monitor the status of cellular gene expression without gene recombination in a non-destructive manner17-26. Although MB has shown great potential for detecting specific target mRNA in vitro, it is often digested by nucleases in vivo, making the lifetime of MB in living cells short. Nanoparticle-based technique that encapsulates MBs and keeps releasing them elongate their working lifetime in living cells tremendously, and realized the long term monitoring of gene expression status in single cells even through the cellular differentiation for almost 20 days27,28. However, the use of nanoparticles as an artificial substance is suggested to be harmful in some cases29,30 and thus the utilization of this technique still has some limitations for regenerative medicine. The degradation of MB often causes another problem; false positive signals. To avoid this, a dual fluorescence resonance energy transfer (FRET)-based MB system was developed31, 32. In this system, a FRET signal is generated only when two MBs are hybridized to a target mRNA side by side, and thus the rate of false positive signals is dramatically reduced. However, in this dual system, the two MBs and the target mRNA must form a trimolecular complex in a highly condensed intercellular environment, and thus there are concerns that the signal response speed of this mechanism is slow.

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(a)

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fluorescence spectra (see Supporting Information). Subsequently, the ability of the DNA-NTs to recognize the intrinsic target mRNA produced by a cell was confirmed by fluorescence in situ hybridization (FISH) [with the FRET-based DNA-NT for GLUT1 (Figure S-6a and Figure S-7), for GLUT4 (Figure S-6b and Figure S-8), and for Control which is designed with the modification of nonsense MB20,31 (Figure S-1b, Figure S-6c and Figure S-9), respectively]. The FISH analysis revealed the behaviors of GLUT mRNA expression responses after insulin stimulation which roughly agree with the results of the semi-quantitative RT-PCR analysis (Figure 2), supporting the importance of single cell analysis. Also it has been shown that the FRET-based DNA-NT does not produce the FRET signal when it binds indiscriminately to a substance inside a cell; rather, it produces the FRET signal only when it binds to the targeted mRNA, dramatically reducing the false positive signal and enabling target detection without further washing steps. Therefore, it is expected that the dynamics of specific mRNA inside a living cell can be observed with this FRET-based DNA-NT probe. Details for these discussions were described in Supporting Information. (a)

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We have been developing a new DNA-based detection probe for specific target mRNA33,34. A FRET-based DNA nano-tweezer structure (DNA-NT) that uses FRET as the detection signal and functions as a single molecule has been developed for imaging analysis (Figure 1a). This FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. We have previously reported that FRET-based DNA-NTs are able to detect both a target mRNA inside a fixed cell and a target inside a living cell33. However, even in the latter case, the cells were fixed to observe FRET signals after the introduction of DNA-NTs into a living cell. In this report, we discuss the feasibility of continuous monitoring of specific mRNA expression in response to an intercellular signal in living cells with a FRET-based DNA-NT.

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Figure 1. Principle of target mRNA detection with FRETbased DNA-NT and sequences for the FRET-based DNA-NTs used in this study: (a) a FRET-based DNA-NT self-assembled from three single-stranded DNAs alters its structure from an open state to a closed state, producing a FRET signal in response to target mRNA recognition; (b) the structure of DNANT for GLUT133 As a target model, we have selected the expressions of glucose transporters (GLUT) 1 and 4. The insulin signal promotes the expression of GLUT1 mRNA35,36 and suppresses the expression of GLUT4 mRNA37. In particular GLUT1 is one of the major glucose uptake transporters in hepatocytes, which are the major glucose uptake cells under a normal condition38. In addition, GLUT4 protein is known to be the major glucose transporter, the location of which is regulated by the insulin signal39,40. Therefore, we believe that the ability to continuously evaluate changes in the expression of these GLUT genes in response to insulin signals in living cells is important for the diagnosis of diabetes. Materials and Methods The detailed procedures for all experiments are described in Supporting Information. Results and discussion Confirmation of target recognition abilities First, the target detecting capabilities of DNA-NTs for GLUT1 (Figure 1b and S-5a) and GLUT4 (Figure S-1a and Figure S-5b) were confirmed in vitro by measuring the relative

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Figure 2. Relative expression of GLUT mRNAs in insulinstimulated Hepa1-6 cells over time, measured by the conventional semi-quantitative RT-PCR method: (a) GLUT1 mRNA and (b) GLUT4 mRNA. The relative values were calculated in relation to the value at 0 h of the insulin non-stimulated cells. The results in the Figures represent the mean ± standard deviation of three replicates. Confirmation of FRET-based DNA-NT introduction into living Hepa 1-6 cells41,42 As shown in the confocal section images in Figure S-10, a clear fluorescence was observed corresponding to Cy5, which reflects the presence of a critical amount of the FRET-based DNA-NT, confirming the successful introduction of the probes inside a living cell by the treatment of Streptolysin O43. However, unfortunately, almost no fluorescence corresponding to Cy5 signal was observed in a central area, which was assumed to be the nuclei. Although the location of the produced FRET signal provides valuable information for the analysis of mRNA expression responses, as discussed in the FISH analysis (see Supporting Information), the current protocol did not

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Figure 3. Representative live-cell images measured with the FRET-based DNA-NTs introduced in Hepa1-6 cells 0, 2, 4, and 6 h after insulin stimulation: (a) merged differential interference contrast (DIC) and FRET images measured with the DNA-NT for GLUT1; (b) FRET image only measured with the DNA-NT for GLUT1; (c) merged DIC and FRET images measured with the DNA-NT for GLUT4; (d) FRET image only measured with the DNA-NT for GLUT4; (e) merged DIC and FRET images measured with the DNA-NT for Control; (f) FRET image only measured with the DNA-NT for Control. allow the active distribution of the DNA-NTs inside the nusignals in these cases were rather stable. Based on these results, cleus. These results indicate that further modification of the it has been shown that the FRET signals correctly reflected the proposed probe is needed, such as the addition of a nuclear behaviors of GLUT gene expressions in response to insulin. localization signal to the probe. However, because the protein As discussed in case of FISH analysis, the gene expression translation occurs in the endoplasmic reticulum, which is loresponses were different from cell to cell, even when the cells cated in the cytoplasm, it is still expected that the FRET signal were given the same stimulation, indicating the importance of observed in the cytoplasm can be an effective indicator of continuous evaluation of the cellular responses within individcellular activity in response to insulin. ual identical cells. Therefore, regions of interest (ROI) were selected for each single cell (Figure S-14a and Figure S-15a), and the FRET signal observed in these regions was calculated. Continuous monitoring of time-dependent gene expression Figure S-14b, Figure S-14c, Figure S-15b and Figure S-15c in living Hepa 1-6 cells show the FRET signal changes over time from each individual Finally, the FRET signal responses from the DNA-NTs incell, indicating totally different behaviors from cell to cell, troduced in living cells were continuously observed after insueven when detected with the same FRET-based DNA-NT. lin stimulation. As seen in Figure 3a, 3b, and Movie S-1 for Unfortunately, in each case neither a clear tendency nor the GLUT1 mRNA and Figure 3c, 3d, and Movie S-2 for GLUT4 significance of each single cell analysis could be identified at mRNA, the DNA-NTs successfully produced the FRET signal this time. However, the approximate tendency of the integratuntil 6 h after insulin stimulation, whereas no signal was deed FRET signals as a mass behavior appeared to be similar to tected in the control case (Figure 3e, 3f and Movie S-3). In all that indicated by conventional mass analysis using semicases, the introduction of the FRET-based DNA-NTs was quantitative RT-PCR, as discussed above. Moreover, some confirmed by the observation of live-cell fluorescence images cells indicting increasing FRET intensity tended to show the attributed to Cy3 and Cy5 dyes (Figures S-11 to S-13). SubseFRET signals in a round shape which is possibly a shape of quently, we integrated the FRET signals in the live-cell images nucleus (Figure S-16a and S-16b for GLUT1, and Figure Sas the total mass amount of expressed mRNA and normalized 16c and S-16d for GLUT4). This behavior may be explained them by the number of cells in the images. Then we compared as follows; the FRET-based DNA-NTs introduced in cytothe signal behaviors with the results of semi-quantitative RTplasm were captured at the surface of nucleus by target PCR analysis. As is apparent from the Figures, all of these mRNAs exported from the nucleus, and were producing the total mass analyses indicated a similar tendency for both FRET signals. Therefore the cells actively synthesizing target GLUT1 (Figure 4a and Figure 2a) and GLUT4 mRNA (Figure mRNAs were observed to exhibit the FRET signals in a round 4b and Figure 2b) against insulin stimulation. GLUT1 mRNA shape. From these discussions, we believe that each individual continued to increase until 4 h of stimulation and then deanalysis reflects the gene expression behavior of each individcreased, whereas GLUT4 mRNA increased immediately, then ual cell in response to insulin. Although the analytical resoludecreased until 4 h and finally increased again at 6 h. Furthertion, including the signal to noise ratio, should be further immore, the FRET signals measured with both DNA-NTs in the proved, and it is necessary to investigate the detection limit of absence of insulin stimulation behaved differently in that the

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this technique as reported in case of fluorescent-dye-based MB44, the proposed probe will become a more powerful tool to monitor continuous cellular gene expression response without gene recombination. (a)

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AUTHOR INFORMATION Corresponding Author * Hisakage Funabashi [email protected]

Notes The authors declare no competing financial interests.

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ACKNOWLEDGMENT

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We would like to thank Editage (www.editage.jp) for English language editing in the first draft. This work was partially supported by JSPS KAKENHI Grant Number 26289314.

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Figure 4. Normalized integrated-FRET intensity measured from live-cell images plotted as a function of time: (a) measurements from DNA-NT for GLUT1. The numbers of cells that contribute to the integrated-FRET intensities were 18 for insulin stimulated cells, 12 for non-stimulated cells; (b) measurements from DNA-NT for GLUT4. The numbers of cells that contribute to the integrated-FRET intensities were 19 for insulin stimulated cells, and 28 for non-stimulated cells. Squares indicate measurements from the DNA-NTs after insulin stimulation; circles indicate measurements from the DNANTs in the absence of insulin; and triangles indicate measurements from DNA-NT for Control after insulin stimulation (integrated from 17 cells and commonly used for each figure). Conclusion In this report, the feasibility of live-cell monitoring of GLUT1 and GLUT4 mRNA expression utilizing FRET-based DNA-NTs has been discussed. The behaviors of the integrated FRET signals of each individual cell in response to insulin stimulation indicated trends similar to those measured with the conventional mass analysis using semi-quantitative RT-PCR. Therefore, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with the proposed FRET-based DNA-NT even in individual cells. Although further development is required to apply this method for diagnostic proposes such as biopsy, the proposed method offers a new approach for living cellular diagnostics based on the evaluation of gene expression response against stimulation. ASSOCIATED CONTENT Supporting Information Additional information and discussion is included, as noted in the main text. This material is available free of charge via the Internet at http://pubs.acs.org.

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