Molecular Switching of a Self-Assembled 3D DNA Nanomachine for

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Molecular Switching of Self-Assembled 3D DNA Nanomachine for Spatiotemporal pH Mapping in Living Cells Yu-Jie Zhou, Yuan-Hui Wan, Cun-Peng Nie, Juan Zhang, Ting-ting Chen, and Xia Chu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02514 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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Analytical Chemistry

Molecular Switching of Self-Assembled 3D DNA Nanomachine for Spatiotemporal pH Mapping in Living Cells Yu-Jie Zhou, Yuan-Hui Wan, Cun-Peng Nie, Juan Zhang, Ting-Ting Chen* and Xia Chu* State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China ABSTRACT: DNA nanomachines have received great interesting due to their potential to mimic various natural biomolecular machines. Intracellular pH sensing and imaging are of great significance to understand cellular behaviors and disease diagnostics. In this work, we report a novel molecular switching of self-assembled 3D DNA triangular prism nanomachine (TPN) for pH sensing and imaging in living cells. The TPN was self-assembled in quantitative yields by hybridization with two DNA triangles and three I-strands (contained i-motif sequence). At the acidic condition, the TPN was compressed due to the I-strand was formed an intramolecular i-tetraplex, which closed proximity between the fluorophores Cy3 and Cy5, resulting in a significant fluorescence resonance energy transfer (FRET) signal. At the neutral or weak alkaline condition, the TPN adopted an extended state due to the random coil form of I-strand, leading to the spatial separation of two fluorophores and the FRET was blocked. The TPN was fully reversible and could rapidly response the pH changes, entered to living cells automatically via an endocytic pathway, monitored spatiotemporal pH changes during endocytosis, maintained its structural integrity after escape from lysosomes and still had the ability for pH sensing, and also visualized pH fluctuations under varying stimuli in living cells. We foresee that this TPN can become a generic platform for pH-related cell biology study and disease diagnostics.

switched flexibly and gained reversible fluorescence resonance energy transfer (FRET) signal, thus effectively enhanced the contrast and accuracy in fluorescence imaging. However, these DNA nanomachines used to require modification with receptor molecules or transfection agent to facilitate them to enter living cells, and some work was even performed by using fixed cells, and had poor stability in complex environment. Some pH sensors based on nanoparticles assembled with i-motif strand have also been reported for intracellular pH sensing.26,27 Although these sensors exhibited efficient cellular uptake, they were not realize the intracellular pH reversible sensing. Studies have revealed that self-assembled 3D DNA nanostructures could maintain structural integrity in complex biological systems, and enable to across the cell membrane through nonspecific uptake.28,29 Moreover, the 3D DNA nanostructures could be constructed with various functional module in a predictable manner by Watson-Crick base-paired interaction.30 Therefore, development of novel 3D DNA nanomachines constructed with pH sensitive DNA sequences and formed molecular switching of pH response, to enter to living cells automatically, for fully reversible and rapidly response the pH changes within living cells, would be highly desirable. Herein, we report a molecular switching of self-assembled 3D DNA triangular prism nanomachine (TPN) for pH sensing and imaging in living cells. This TPN was able to probe pH fluctuations under external stimuli in living cells and monitor spatiotemporal pH variations during endocytosis. The TPN was self-assembled in steps according to the previous strategies.31-32 As illustrated in Scheme 1A, three DNA strands, which possessed central region a* or b*, brought together to achieve the triangles (named T1 and T2) by hybridization with two 15-base sequences at either side of each strand. The I-

DNA nanomachines, a variety of self-assembly structures by artificial design, can move and change conformation upon stimulation by external triggers.1 In the last ten years, DNA nanomachines have received great interesting due to their potential to mimic various natural biomolecular machines.2 A lot of DNA nanomachines have successfully applied in biological applications, including biosensor,3,4 logic computing,5 autonomous motor,6 cargo-sorting7 and regulation of cell function.8 Although some DNA nanomachines have been demonstrated for sensing intracellular molecules, they still need cellular delivery systems, such as transfection agent,9 aptamer-assissted,10 and nanocarrier.11 Therefore, development of novel DNA nanomachines without any ancillary delivery systems and operated in living cells remains very required. Self-assembled 3D DNA nanostructures have an ability to enter cells, exhibited intrinsic biocompatibility, low immunogenicity, highly programmability, nearly quantitative yields, stability towards nuclease susceptibility,12-15 and were successfully applied in sensing,16 drug carriers,17,18 and cargo encapsulation.19 However, 3D DNA nanostructures that switched molecular conformations in reversible response to microenvironment stimulation in living cells, have been still unexplored. Intracellular pH is a crucial parameter associated with various cellular behaviors and biological processes.20 Abnormal pH represents a typical feature of many diseases, such as cardiopulmonary and neurological diseases, diabetes, myocardial ischemia, and even cancers.21 Therefore, intracellular pH sensing and imaging are of great significance to understand cellular behaviors and disease diagnostics.22 Among numerous pH sensors, self-assembled DNA nanoprobes, including nano-tweezer,23 intermolecular triplex DNA24 and i-motif,25 have great advantages that can be 1

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strand included three regions, region a and b were labeled with Cy3 at 3’ end and Cy5 at 5’ end, respectively. Region c was an i-motif sequence that could form an i-tetraplex consisting of two parallel-stranded C-H.C+ base-paired duplexes under acidic condition.33 The TPN was obtained by mixing one of T1, one of T2, and three of I-strand, via the regions of a and b in Istrand complementary to the regions of a* and b* in the two triangles. When the TPN entered the cell and localized in the lysosomes, acidic compartments in living cells,34 the i-motif sequences in TPN were protonated, and folded to form an intramolecular i-tetraplex, which closed proximity between the fluorophores Cy3 and Cy5, resulting in a significant FRET signal. When the TPN end up in the cytoplasm, the neutral pH condition in living cells,34 the TPN adopted an extended state due to the random coil form of I-strand, resulting in spatial separation of the two fluorophores and the FRET was blocked (Scheme 1B). In this work, we report a novel pH sensing platform based on 3D DNA triangular prism nanomachine. Compared with other pH-responsive DNA probes, this DNA nanomachine could enter to living cells automatically, provide more fluorescence signals to enhance sensing contrast, maintained its structural integrity after escape from lysosomes and still had the ability for pH sensing. The TPN give an ideal platform for a high contrast in spatiotemporal pH mapping during endocytosis and intracellular pH sensing.

DNA TPN (Figure S3) and demonstrated the successful assembly of the TPN. Further characterization of TPN was performed using circular dichroism (CD) spectroscopy. The CD data confirmed that both the TPN and single I-strand exhibited the same changes of CD phenomenon under various pH values (Figure S4), suggesting that the configuration of TPN did not influence the i-motif sequence to pH response. The gel electrophoresis analysis of the TPN at different pH buffers (7.5 and 5.0) revealed that the TPN in pH 5.0 migrated faster than that in pH 7.5 (Figure S5), which might attribute to the compression of TPN induced by the i-motif folding at acidic condition, resulting the size of TPN in pH 5.0 buffer is smaller than that in 7.5 buffer. We chose the fluorophores Cy3 and Cy5 as donor and acceptor for FERT system respectively, because their fluorescence intensity would not change in different pH solutions (Figure S6). Next, we explored the fluorescence spectra of the TPN and single I-strand at different pH buffers. As shown in Figure 1B, when the TPN was in pH 7.5 buffer, the FRET was blocked. The fluorescence spectra of the TPN exhibited an increased Cy3 fluorescence at 562 nm and decreased Cy5 fluorescence at 664 nm (the ratio of intensity from Cy3 to Cy5 was 1.98). However, at pH 5.0, a significant FRET signal was observed (the ratio of intensity from Cy3 to Cy5 was 0.53). For single I-strand, the Cy3 fluorescence at 562 nm was obviously decreased but Cy5 fluorescence at 664 nm was little increased when the pH changed from 7.5 to 5.0, corresponding with the ratios were changed from 1.32 to 0.20. The distinct FRET efficiency may be associated with the more signals in one TPN molecule and the random swing of the region a and b in the I-strand.

Scheme 1. (A) Illustration of the TPN Assembly; (B) Illustration of the TPN for Spatiotemporal pH Mapping and Sensing in Living Cells.

We first demonstrated that the TPN was self-assembled in steps by agarose gel electrophoresis analysis. The triangles T1 and T2 were assembled by mixing three complementary strands with a concentration ratio at 1:1:1, at 37 °C for 30 min, respectively (Figure S1, lane 3 and lane 6). The TPN was progressively formed by mixing T1, T2, and I-strand with a concentration ratio at 1:1:3, at 37 °C for 3 hours. There were not any other polymeric substances generated, which confirmed the TPN was obtained in quantitative yields (Figure 1A, lane 8), indicating that the concentration of TPN could be calculated by stoichiometric ratio. The atomic force microscopy (AFM) characteristic demonstrated that our TPN possessed good dispersity with mean sizes of 15.2±2.1 nm (Figure S2A). The DLS study also gave a narrow size distribution of the TPN with an average size of 13.6±2.0 nm (Figure S2B). These results were in good agreement with the theoretical estimate of 9.4 nm for the diameter of the rigid

Figure 1. (A) Gel electrophoresis of the formation of TPN. Lane 1, T1-1; lane 2, T1-1 and T1-2; lane 3, T1; lane 4, I-strand; lane 5, T1 and I-strand (1:1); lane 6, T1 and I-strand (1:2); lane 7, T1 and I-strand (1:3); lane 8, TPN. (B) The fluorescence spectra of the TPN and single I-strand at the different pH buffers. (C) The reversibility of TPN and single I-strand against pH change between pH 7.5 and 5.0, repeatedly. (D) The plot of the ratio (ICy3/ICy5) of fluorescence intensity of the TPN versus pH values. The error bars are the standard deviations of three parallel tests.

A further reversibility study of the TPN and single I-strand were performed by adjusting the samples between pH 5.0 and pH 7.5. Both the TPN and I-strand showed excellent reversibility for several pH cycles without obvious attenuation in different pH solutions (Figure 1C). We also observed that abrupt changes in pH environment leaded to fast changes in the fluorescence ratios of the TPN and single I-strand (Figure 2


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Analytical Chemistry with the acidic organelles, which was stained by LysoTracker, and the Pearson’s coefficient (calculated according to the fluorescence of Cy3 and LysoTracker) changed from 0.10 (30 min) to 0.37 (60 min) and then 0.56 (90 min). These results revealed that the TPN could traffic into the acidic organelles. To our surprise, with the incubation time increasing, the fluorescence intensity in Cy3 channel became stronger, and the ratio imaging of Cy3 to Cy5 changed visibly from multicolor (120 min) to mainly red (180 min) in the white frames, while the Pearson’s coefficient changed from 0.32 (120 min) to 0.24 (180 min), suggesting that the TPN was partly conveyed from acidic to neutral environment. These results indicated that the TPN could ultimately partly escape from acidic endo-lysosomes into the neutral cytoplasm. The higher resolution images of the Cy5-labeled TPN and another DNA triangular prism with ploy-T sequences instead of imotif sequences (TPN-T), gave much clearer evidence that the TPN itself was able to partly escape from endo-lysosomal into cytoplasm (Figure S13). Moreover, the changes of the ratio of Cy3 to Cy5 in TPN were correlated with the pH changes in different spatiotemporal distribution during the endocytosis process. As shown in Figure S14, at the first 10 min, the ratios of Cy3 to Cy5 was mainly in the range of 1.5~2.0 (corresponding to a pH range of 6.8~7.4), implying that most TPNs (~95%) mainly adsorbed at the cell membrane. As time prolonged, the pH values of most TPNs gradually changed from 7.4 to 5.6~6.2 range at 30 min, and further changed to 5.0~5.6 range at 90 min. This phenomenon indicated that most TPNs internalized into endo-lysosome compartment through endocytosis when incubated for 90 min. Subsequently, as time further prolonged, pH of most TPNs changed gradually from

S7). These results implied that the TPN had the same rapid pH response ability as the single I-strand. We then investigated the detection capability of the TPN and single I-strand for pH sensing, respectively. Both in TPN and single I-strand, sequentially decreased in Cy3 signal (562 nm) and gradually increased of Cy5 signal (664 nm) were observed with decreasing pH in solutions (Figure S8 and Figure S9A). Compared with single I-strand, the TPN presented excellent linear to the pH response and the range was from 5.4 to 6.4 (Figure 1D and Figure S9B). This linear range covered the physiological pH values of the living cells and indicated that the TPN promisingly afforded a useful sensor for pH sensing and imaging in living cells. The major challenge of using DNA nanostructures for their applications in cell biology was the stability of the DNA nanostructures in complex biological conditions, in which the low Mg2+ concentration and the digestion by nucleases would result in the degradation of DNA nanostructures.35 To confirm the stability of our nanomachine, the TPN was incubated with different biological conditions at 37 °C for different time periods, including culture medium, DNase I and cell lysates. As shown in Figure S10, the bands of the TPN remained clear after incubation in different biological conditions, however, the bands of the single I-strand were gradually blurred when incubated with culture medium or 0.25 U/mL DNase I, and almost completely disappeared in the cell lysates. These results demonstrated that the TPN possessed better stability in complex biological conditions compared with single I-strand, and the 3D DNA nanomachine could protect oligonucleotides from nucleases-mediated degradation and maintain structural integrity in cell culture media. Before studying the operation of the TPN in living cells, we investigated the toxicity of TPN. As shown in Figure S11, the TPN exhibited only marginal toxicity to HeLa cells, even at a concentration as high as 400 nM, and the cell viability decreased by only ~15% after 8 h incubation. These results revealed that the TPN exhibited good biocompatibility. To prove that the TPN exhibited efficient cellular uptake, we prepared the self-assembly TPN (66.7 nM) and incubated with HeLa cells in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C. After 3 hours of incubation, the cells displayed obvious fluorescence both in Cy3 channel and Cy5 channel (Figure S12). In contrast, the single I-strand (200 nM) was directly incubated with the cells, and no appreciable fluorescence was obtained in Figure S12. These results revealed that the TPN could enter to living cells automatically. We first studied the internal existence of the TPN in the process of endocytosis. Cells were incubated with 66.7 nM TPN for distinct durations, and fluorescence images were then taken after staining of acidic organelles using LysoTracker Green DND-26. As shown in Figure 2, by 10 min incubation, most of the TPN were on the cell surfaces and showed bright green fluorescence and negligible red fluorescence. At this time, no apparent colocalization of TPN with LysoTracker was observed, revealing that the TPN was in an extracellular neutral environment. With time progressing, lots of the TPN were internalized into the cells, and the fluorescence intensity in Cy5 channel became stronger. The ratio imaging of Cy3 to Cy5 (the fifth column in Figure 2) changed apparently from mainly green (30 min) to partial blue (60 min) and then almost purple (90 min) in the white frames, indicating that the FRET signal from Cy3 to Cy5 of the TPN was increased over time. During this time, the TPN was found partial colocalization

Figure 2. Ratiometric fluorescence images of mapping spatial and temporal pH changes of HeLa cells treated with TPN at the indicated times of 10, 30, 60, 90, 120, and 180 min, respectively. Scale bar: 10 μm. 3



5.0~5.6 to 7.4, implying that most TPNs (~60%) escaped from endo-lysosome compartment to cytoplasm and the TPN could be still working but not broken even the FRET signal was recovered. These pH values in different microenvironments were consistent with previous report.36 These results revealed that the TPN could enter to living cells automatically, partly released from lysosomes into cytoplasm, and gave clear evidence for mapping spatiotemporal pH changes during endocytosis, which could not be achieved with many other DNA-based nanomachines. Next, we studied the potential of the TPN for pH sensing and imaging in living cells. HeLa cells were incubated with 66.7 nM TPN at 37 °C for 3 h, followed by treatment with 15 μM H+/K+ ionophore nigericin, which could homogenize the concentration of H+ in the whole cells to the surrounding medium.21 As shown in Figure S15, when the cells were incubated with pH 5.0 buffer, negligible green fluorescence and bright red fluorescence (FRET signal) were obtained, indicating that the TPN was compressed. In contrast, after incubation with another pH 7.4 buffer, the cells gave increased green fluorescence and dim red fluorescence, suggesting that the TPN adopted an extended state in weak alkaline pH condition. Interestingly, reincubation of the cells with pH 5.0 buffer, recovered strong FRET signal in the cells. But the cell morphology was affected when measuring pH reversible conversion. These results validated that the TPN was fully reversible molecular switching of pH sensing in living cells. We further explored the application of TPN for imaging of living cells with different pH values. We observed that the fluorescence intensity of Cy3 was sequential increased while Cy5 was gradually decreased with increasing pH (Figure 3). The ratio changes (Cy3/Cy5) of the TPN (Figure S16) were also correlated with different pH values in living cells. These results implied that the TPN had the ability to monitor pH change in living cells.

cells were treated with 100 µM NaClO, the fluorescence signals in two channels were not obviously changed, suggesting that the elevated level of ClO- could not increase intracellular acidic, which was also proved by the previous report.37 The corresponding ratio values of Cy3 to Cy5 were showed in Figure S15B. As a result, the TPN offered a useful sensor for visualizing pH fluctuations under external stimuli in living cells. In conclusion, we had successfully developed a molecular switching of self-assembled 3D DNA triangular prism nanomachine (TPN) for intracellular pH sensing and imaging. The TPN had the properties of easy and rapid self-assembly, full reversibility, high contrast and accuracy pH-responsive in physiological pH ranges. Moreover, compared to existing pHresponsive DNA probes, the TPN exhibited efficient cellular uptake via an endocytic pathway, monitored spatiotemporal pH changes during endocytosis, maintained its structural integrity after escape from lysosomes and still had the ability for pH sensing, and also visualized pH fluctuations under varying stimuli in living cells. Therefore, our TPN can become a generic platform for pH-related cell biology study and disease diagnostics.

ASSOCIATED CONTENT Supporting Information Experimental details and additional figures as noted in the text. This material is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Phone: 86-731-88821916. Fax: +86-731-88821916.; E-mail: [email protected], [email protected]; Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grants 21525522 and 21705039), the National Postdoctoral Program for Innovative Talents (BX201600049).

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Figure 3. Ratiometric fluorescence images of the TPN for nigericin-treated HeLa cells at pH 5.0, 5.6, 6.2, 6.8 and 7.4 PBS buffer, respectively. Scale bar: 20 μm.

We also used the TPN to explore the effect of reactive oxygen species on pH fluctuation in living cells. As shown in Figure S17, there was weak FRET signal in untreated cells. After treated with 100 µM H2O2, bright Cy5 fluorescence and dim Cy3 fluorescence were exhibited in cells. This result implied that the intracellular pH was dropped sharply due to the production of acidic substances by oxidative stress, which was consistent with the previous work.37 However, when the 4


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Molecular Switching of a Self-Assembled 3D DNA Nanomachine for

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