Cell Surface-Anchored DNA Nanomachine for Dynamically Tunable

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Cell Surface Anchored DNA Nanomachine for Dynamically Tunable Sensing and Imaging of Extracellular pH Lan Liu, Cai-Xia Dou, Jin-Wen Liu, Xiang-Nan Wang, Zhan-Ming Ying, and Jian-Hui Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03589 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

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Analytical Chemistry

Cell Surface Anchored DNA Nanomachine for Dynamically Tunable Sensing and Imaging of Extracellular pH Lan Liu, Cai-Xia Dou, Jin-Wen Liu, Xiang-Nan Wang, Zhan-Ming Ying, Jian-Hui Jiang* Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/ Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China * Corresponding Author: Fax: +86-731-88821916; E-mail: [email protected]

ABSTRACT: DNA nanodevices that mimic natural biomolecular machines changing configurations in response to external inputs have enabled smart sensors to live cell imaging. We report for the first time the development of a dynamic DNA nanomachine that is anchored on cells surface and undergoes pH-responsive triplex-duplex conformation switching, allowing tunable sensing and imaging of extracellular pH. The results reveal that the DNA nanomachine can be stably anchored on cell surface via multiple anchors, and the adjustment of C+G-C content in the switch element confers tunability of pH response windows. The anchored DNA nanomachine also demonstrates desirable sensitivity, excellent reversibility and quantitative ability for extracellular pH detection and imaging. This cell-surface-anchored pH-responsive DNA nanomachine can provide a useful platform for pH sensing in extracellular microenvironments and diagnostics of different pH-related diseases.

Living organisms comprise various biomolecular machines capable of undergoing conformational changes in response to environmental stimuli.1 The exquisite regulation of biomolecular machines have motivated extensive efforts to mimic biological activities using artificial nanodevices.2-4 In the context, DNA have offered unique advantages for constructing various dynamic nanodevices in a predictable manner using their specific Watson-Crick interactions.5 With combination of versatile modifications of reactive and reporting groups,6 or functional DNA modules such as DNA probes,7 DNA triplex,8 aptamers,9 and DNAzyme,10, DNA nanodevices can been engineered to mimic various biomolecular machines,11 including autonomous walkers,12 13 14 nanotweezers and nanoscissors. Most of current DNA nanomachines have been demonstrated for in vitro applications.15 Very recently, DNA nanomachines have been applied to sensing intracellular molecules16 and transient encountering events.17 However, artificial DNA machines that are anchored on specific subcellular localization in living cells for microenvironment sensing and regulation have been largely unexplored. Abnormal extracellular pH is known to be associated with various pathological states, such as those in tumors, ischemic stroke, infection and inflammation.18 Specifically, dysregulated pH is regarded as a hallmark of cancer, because extracellular pH surrounding cancer cells is lower than that inside the cells, which is contrary to the pH gradient in normal cells.19 Hence, sensing of pH near the cell surface is of great importance to diagnosis and understanding of these diseases.20-22 Along the direction, DNA sensors has been designed for detection and

imaging of extracellular pH based on specific organic fluorophores labeled to DNA probe21 or pH-responsive i-motif DNA22. However, these sensors are typically tethered on cell surface using one phospholipid or cholesterol moiety, showing poor stability and substantial leakage from cell surface. Moreover, the dynamic response ranges of the sensors are exactly determined by the i-motif DNA and fluorophores, which are difficult to be finely tuned for applications with varying pH ranges. Therefore, engineering of novel DNA sensors allowing stable anchoring on cell surface and dynamically tunable sensing of extracellular pH remains a challenge. Scheme 1. Illustration of cell-surface-anchored nanomachine as reversible and tunable pH sensor.

DNA

Here we develop a novel DNA nanotweezer (NT) sensor with triplex-regulated conformational switches,23 which enables stable anchoring on cell surface and

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dynamically tunable detection and imaging of extracellular pH, as illustrated in Scheme 1. It is demonstrated that sequence-selective recognition of a DNA duplex at the major groove by a third strand through Hoogsteen hydrogen bonds can form triplex structures.24 The formation of C+-G-C triplets is pH-dependent due to the need for protonated cytosine in the triplex.24 Moreover, with different percentage of C+-G-C triplets, the pH-responsiveness of DNA triplexes can be programmed, affording possibility of tuning the dynamic range in pH detection.25 Motivated by the rationale, we develop a DNA NT that is constructed using three DNA strands (F, L or L2, and S or S2) with the triplex-forming sequences (L or L2, and S or S2) designed with varying percentage of C+-G-C triplets. This design affords the DNA NT with duplex-triplex conformational switching in response to pH with tunable dynamic ranges. To deliver a fluorescence signal indicating the conformation changes of the NT, a fluorophore TAMRA and a quencher BHQ2 are labelled at two ends in strand F (FnQ-F). Furthermore, to stably anchor the NT on cell surface, we designed each DNA strand with a covalent modifier of cholesterol (Chol) via a triethyleneglycol spacer. Thus, a DNA NT with three Chol modifiers can be prepared with three Chol-modified stands (Chol-F, Chol-L or Chol-L2, and Chol-S or Chol-S2) (Figure S1). When Chol-modified NT incubated with cells, its hydrophobic cholesterol units enable the NT to insert into the hydrophobic interior of lipid membranes through the hydrophobic interaction,17, 21, 26 and the hydrophilic DNA NT unit extends out from the membrane to sense the extracellular pH. According to the design, the NT will take the closed state in cases when triplex is form between strands S and L under acidic extracellular microenvironments, generating a quenched fluorescence signal because of the close proximity between the fluorophore and quencher. At basic extracellular microenvironments, the triplex dissociates and the NT is opened by the duplex stretch regions formed between S and L as well as F and L. This opened state renders an activated fluorescence because the fluorophore is drawn apart from the quencher. Because the triplex formation is rapidly and reversible responsive to pH, the NT is then capable of monitoring in real time the extracellular pH changes. To our knowledge, it is the first time that a DNA nanomachine constructed by multiple DNA sequences has been anchored on cell surface for extracellular microenvironment sensing. Unlike current cell surface anchored sensors using DNA single or double strands,21,22 DNA nanomachines afford a new biosensor scaffold with highly programmable structures and site-specific modifications. The construction of DNA nanomachine using multiple strands provides multiple modification sites for anchoring moieties. This multi-site membrane anchoring strategy allows efficient and stable tethering of the sensor on cell membranes, and also minimizes detachment of the sensor from cell membrane.27 Moreover, the DNA nanomachine enables programmable

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responsiveness through regulating the percentage of C+-G-C triplets in the triplex domain.25 This design affords fine tunability of the dynamic ranges in pH detection. Therefore, our nanomachine can provide a useful platform for pH sensing in extracellular microenvironments and diagnostics of different pH-related diseases.

Figure 1. A) Confocal images at high magnification for CCRF-CEM cells with surface anchored TAMRA-3Chol-NT and nuclei stained with Hoechst 33258. B) Corresponding images at low magnification. C) Flow cytometry data of cells. 1) CCRF-CEM cells, 2) cells incubated with TAMRA-1Chol-NT, 3) cells incubated with TAMRA-2Chol-NT, 4) cells incubated with TAMRA-3Chol-NT, 5) cells incubated with TAMRA-3Chol-NT followed by DNase I treatment, 6) cells incubated using TAMRA-NT without Chol anchor. D) Flow cytometry profiles of cells incubating with TAMRA-NT with varying Chol anchors after centrifuging for different times.

To demonstrate the feasibility of anchoring the DNA NT on cell membrane, we synthesized NTs labeled with a TAMRA fluorophore and a varying number (1 to 3) of cholesterol moieties (TAMRA-1Chol-NT, TAMRA-2Chol-NT, and TAMRA-3Chol-NT). Because anchoring of Chol- or lipid-modified DNA probes on cell membrane could be efficiently accomplished at 4 °C, and such a incubation condition also minimized cellular endocytosis, we chose to incubate the TAMRA-3Chol-NT with CCRF-CEM cells at 4 °C. After 20 min incubation and Hoechst 33258 staining, the cells showed bright orange (pseudocolor in red) fluorescence primarily localized on cell membrane (Fig 1A and B). Further z-stack projection analysis confirmed typical localization of the fluorescence signals on cell surface in all z-axis sections (Figure S2). These data gave clear evidence for the ability of Chol moieties to anchor the NT on cell surface. Flow cytometry analysis revealed that cells incubated with TAMRA-3Chol-NT gained significant fluorescence enhancement compared with the blank cells (Figure 1C). Moreover, the NT with more Chol anchors exhibited higher fluorescence signals on the cells, indicating that multiple Chol anchors allowed more NTs to be tethered on cell surface. A further investigation of the NT-tethered cells treated with 20 U/mL DNase I showed almost


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Analytical Chemistry diminished fluorescence on the cell surface. Because DNase I could not enter the cells and merely cleaved DNA on the cells, this result indeed testified the localization of NTs on the outer cell surface. A control experiment using TAMRA-labeled NT with no cholesterol anchor showed no fluorescence signals on the surface, implying that cholesterol anchors were essential for tethering the NT on cell surface. Closer interrogation of the time-dependent fluorescence signals revealed that anchoring of TAMRA-3Chol-NT proceeded very quickly, substantial loading of the NTs on cell surface appearing after 1 min and saturated anchoring of the NTs finished after 5 min (Figure S3). Interestingly, we found that the NT with more Chol anchors showed improved stability on cell surface. The cells tethered with TAMRA-1Chol-NT showed a slight decline of fluorescence signals after 15 min and 30 min incubation (Fig S4). A smaller decrease of fluorescence was observed for cells tethered with TAMRA-2Chol-NT, and no decrease of fluorescence signals appeared for cells tethered with TAMRA-3Chol-NT. We further challenged the systems by centrifuging (Figure 1D). We observed a gradual loss of fluorescence when TAMRA-1Chol-NT was subjected to once and twice treatment of centrifuging and suspension in a fresh medium. A smaller loss of fluorescence signals was observed for cells tethered with TAMRA-2Chol-NT after the treatment, indicating that two anchors increased the stability of DNA NTs on cell surface. Three anchors were found to further increase the stability of DNA NTs on cell surface, with cell-anchored TAMRA-3Chol-NT almost showing negligible loss of fluorescence after the treatment. These results implied that the multi-site anchoring strategy increased the stability and prevented detaching of the DNA NTs from cell surface. Furthermore, the cytotoxicity of the sensor was tested. We observed that the TAMRA-3Chol-NT only exhibited marginal toxicity to CCRF-CEM cells with the cell viability decreased by ∼8% after 24 h incubation (Figure S5), suggesting high biocompatibility of the NT sensor. Besides, we inspected the universal applicability of the NT sensor for other cell lines including HeLa, MCF-7 and HepG2. Confocal images revealed clear anchoring of the NTs on the plasma membranes of different cells, demonstrating generality of the NT sensor for cell membrane anchoring of different cells (Figure S6). In addition, incubation of the cell-surface anchored NT sensor at 37 oC for 1 h showed only slight endocytosis (Figure S7), implying the potential of the NT sensor for extracellular pH imaging under physiological conditions. Gel electrophoresis was used to investigate the pH sensing performance of the DNA NT (Figure S8). Interestingly, the DNA NT (lane 6) displayed varied electrophoretic mobility at different pH values with slightly slower mobility at pH 8.0 and faster mobility at pH 5.0. This result indicated that the NT had a compactly packed conformation pH 5.0 while took a loosely packed conformation at pH 8.0. This conformation characteristic was ascribed to the formation and dissociation of the triplex in the NT at pH 5.0 and 8.0, respectively. The

conformational changes of the NT were further examined by fluorescence assay using a DNA NT synthesized with L, S and fluorescence-quenched F (FnQ-F). We observed that incubation of the FnQ-NT at pH 5.0 merely gave a weak fluorescence peak (Fig 2A), an indicator of substantially quenched fluorescence of TAMRA. This result suggested a compact conformation or the closed state for the FnQ-NT. In contrast, intense fluorescence was obtained at pH 8.0 with a peak intensity ~4.0-fold higher than that at pH 5.0, implying that the FnQ-NT was in the open state. A control experiment using the NT merely labelled with TAMRA (TAMRA-NT) with no quencher showed only a marginal change in the fluorescence spectra at these two pH values (5.0 and 8.0). This result evidenced that the fluorescence responses was specifically attributed to the distance change between TAMRA and BHQ2, confirming conformation changes of the NT at different pH values. Cyclic study showed that the NT responded reversibly to pH changes (Figure 2B), implying its ability as a reversible pH sensor. Moreover, fluorescence responses of the NT exhibited dynamic and sensitive changes in the pH range from 4.4 to 6.0 with an estimated pKa ~5.5 and reached a plateau at pH 6.0 (Figure 2C and D). Note that this NT was designed to contain 78% C+-G-C triplets in the triplex domain. This high content of C+-G-C triplets gave a DNA NT with pH responsiveness in the acidic pH range (4.4-6.0).

Figure 2. A) Fluorescence spectra of FnQ-NT and TAMRA-NT at pH 8.0 and 5.0. B) Fluorescence intensity changes at 580 nm for FnQ-NT between pH 5.0 (a) and 8.0 (b). C) Fluorescence spectra of FnQ-NT at different pH values. D) Response curves of FnQ-NT and FnQ-NT2 to varying pH values.

Because of the programmability of DNA triplex, we could regulate the pH responsiveness of the NT sensor. Actually, by constructing another DNA nanomachine, NT2, using L2, FnQ-F2 and S with a low content of C+G-C triplets (47%),23 the sensor exhibited dynamic responses in a basic pH window from 6.0-7.7 with an estimated pKa ~7.4 (Figure 2D and S9). We could infer that a higher percentage of C+-G-C triplets in the triplex motif led to a more acidic response window with a lower pKa. This result manifested that pH responsiveness of the NT could be rationally tuned to a specific pH window by designing the triplex-forming domain with varying contents of



C+-G-C triplets. This was indeed a salient advantage of the our triplex-based NT for pH sensing, which could be finely tuned and adapted for applications with varying pH ranges, such as pH sensors in different cells or different organelles of mammalian cells.

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microenvironments of cancer cells, showed fluorescence decreased signals. This defect could be improved by using fluorescence donor-acceptor pair in place of the flurophore-quencher pair in the NT. In general, the cell-surface-anchored DNA nanomachine could provide a useful platform for pH sensing in extracellular microenvironments and diagnostics of different pH-related diseases.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publication website at http://pubs.acs.org. Materials, instruments and methods, DNA sequences, TOF-MS spectra, Z-Scanning confocal images, flow cytometry assay, cytotoxicity assay, confocal images of different cells, PAGE analysis, fluorescence spectrum. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +86-731-88821916. ORCID Jian-Hui Jiang: 0000-0003-1594-4023 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by NSFC (21527810, 21521063).

Figure 3. A) Fluorescence images of FnQ-3Chol-NT anchored on CCRF-CEM cells in media of varying pH values. B) Fluorescence images of FnQ-3Chol-NT2. 1) Overlay images, 2) fluorescence images. C) Fluorescence intensity versus extracellular pH of FnQ-3Chol-NT. D) Fluorescence intensity versus pH of FnQ-3Chol-NT2. Next, we investigated the potential of cell membrane anchored FnQ-3Chol-NTs for extracellular pH imaging on living CCRF-CEM cells. With increasing extracellular pH, both NTs displayed gradual increases of fluorescence brightness on the cell membrane (Figure 3A and B). This result revealed the ability of the developed NTs for sensing extracellular pH in living cells. A dynamic correlation between fluorescence intensity on cell membrane and extracellular pH were obtained (Figure 3C and D). Interestingly, the response curves obtained for cell-surface anchored NTs with confocal microscopy had the same dynamic ranges as those obtained with free NTs in solution using fluorescence assay. This result indicated that surface-anchored NTs had the ability for quantitative detection of extracellular pH in living cells. In conclusion, we reported for the first time the development of a cell-surface-anchored DNA NT for dynamically tunable sensing of extracellular pH on living cells. The DNA NT could be efficiently and stably anchored on cell surface via multi-site cholesterol labels. The results revealed that the cell-surface-anchored DNA NT exhibited reversible conformational changes in response to pH and showed controllable tunability, high sensitivity and quantitative ability for extracellular pH detection. A possible defect of current design was that the NT sensor, when applied to detecting acidic extracellular

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Cell Surface-Anchored DNA Nanomachine for Dynamically Tunable

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