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Proton-Fueled, Reversible DNA Hybridization Chain Assembly for pH Sensing and Imaging Lan Liu, Jin-Wen Liu, Zhi-Mei Huang, Han Wu, Na Li, Li-Juan Tang, and Jian-hui Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01843 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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Proton-Fueled, Reversible DNA Hybridization Chain Assembly for pH Sensing and Imaging Lan Liu, Jin-Wen Liu, Zhi-Mei Huang, Han Wu, Na Li, Li-Juan Tang*, and 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]; [email protected]

ABSTRACT: Design of DNA self-assembly with reversible responsiveness to external stimuli is of great interest for diverse applications. We for the first time develop a pHresponsive, fully-reversible hybridization chain reaction (HCR) assembly that allows sensitive sensing and imaging of pH in living cells. Our design relies on the triplex forming sequences that form DNA triplex with toehold regions under acidic conditions and then induce a cascade of strand displacement and DNA assembly. The HCR assembly has

shown dynamic responses in physiological pH ranges with excellent reversibility and demonstrated the potential for in vitro detection and live-cell imaging of pH. Moreover, this method affords HCR assembles with highly localized fluorescence responses, offering advantages of improving sensitivity and better selectivity. The protonfueled, reversible HCR assembly may provide a useful

approach for pH-related cell biology study and disease diagnostics.

Biomolecules such as proteins, lipids and nucleic acids, are often self-assembling into higher-order structures under the control of intermolecular interactions and external stimuli. These assemblies play versatile roles in cell functions, which also inspires researchers to engineer finely-tuned self-assembled biomolecular complex for profound applications.1 As a promising building material, DNA has been demonstrated the capability to fabricate programmable self-assembled structures of different morphologies.2 Hybridization chain reaction (HCR) represents a simple but efficient approach to constructing linear DNA assemblies.3 In HCR, a single-stranded input initiates a cascade reaction between two kinetically trapped hairpin probes via exposed toehold binding and strand displacement. As a long linear polymer of DNA duplex assemblies is generated, HCR affords the potential for enzymatic signal amplification for nucleic acid detection.4 Moreover, by taking advantage of the conformation changes of DNA aptamers or DNAzymes, HCR assembly has been designed to be activated in response to varying targets,5-7 such as metal ions,5 small biomolecules,6 and proteins.7 Since our report on the realization of HCR in-

side living cells for ultrasensitve imaging of mRNA,8 extensive effort has been made in the development of HCR based sensors for intracellular detection.9-12 In living cells, pH modulates many cellular events and plays a pivotal part in various metabolism processes. The abnormal distribution and fluctuation can cause cell dysfunction or even tumorigenesis and metastasis.13 Measurement of intracellular pH distribution and variation is of great importance for cell biology. Hence, many pH sensitive materials have been developed.14-16 Non-DNA-based pH sensors, including organic fluorescent probes,14 fluorescent proteins,15 and nanoparticle,16 have achieved good performance in living cells. However, they suffer from complicated synthesis, poor biodegradability and unfavorable biocompatibility.17 DNA sensors represent an alternative tool for intracellular pH detection. It is well demonstrated that pH is able to modulate reversible structure switching of specific DNA structures such as imotif and triplex-forming sequences.17,18 Based on the mechanisms, pH-responsive DNA nanodevices has been constructed to map pH changes inside living cells.17,18 It has also been reported that HCR assembly can be controlled by pH under in vitro conditions, and this assembly can be partially reversible in response to switches between acidic and basic pH.19 However, current designs of HCR reactions have not enabled fully reversible HCR assembly and disassembly, while precludes the applications for continuous monitoring of pH variations in response to environmental stimuli. Moreover, the HCR assembly has not been realized to be responsive to pH in living cells. Herein we report, for the first time, the development of a pH-responsive, fully-reversible hybridization chain assembly in living cells that allows sensitive sensing and imaging of intracellular pH. The pH-responsive, fullyreversible HCR assembly relies on the triplex-forming sequence (TFS) that is able to form DNA triplex by using the Hoogsteen base pairs at acidic pH.17 It was reported that transient formation of DNA triplexes was able to trigger strand displacement reaction.20 Because strand displacement is the key reaction in HCR, we assume that

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Scheme 1. a) Illustration of proton-fueled, reversible DNA hybridization chain assembly. b) Proposed mechanism for the HCR assembly process at acidic pH. c) Proposed mechanism of the disassembly process at basic pH.

the TFS can also trigger a HCR assembly in response to acidic pH. Motivated by this hypothesis, we develop a pHresponsive HCR assembly based on TFS toehold induced strand displacement, as illustrated in Scheme 1A. The initiator sequence I is designed to have a TFS domain (a**) and a single-stranded sequence (x*) complementary to the stem region of hairpin probe H1 (x*-x). H1 has a loop of TFS domain (b**) and a duplex toehold region (a-a*). H2 has a loop of TFS domain (a**) and a duplex toehold region (b-b*). At acidic pH (Scheme 1b), C bases in the TFS domain (a**) of I are protonated into C+, allowing formation of a triplex with the duplex toehold region (a-a*) in H1. The formed triplex can then mediate strand displacement between two x* regions in I and H1 and open hairpin probe H1 with an exposed tail of b** and x*. The exposed b**, in turn, forms a triplex with the duplex toehold b-b* in H2, which triggers another toehold-mediated strand displacement by displacing x* in H2 by the exposed x* in the I-H1 assembly. The reaction produces the assembly of I-H1-H2, releasing a** and x* in H2 which can act as the initiator again to continue a successive assembly with H1 and H2. To deliver a fluorescence signal indicating the HCR assembly process, a fluorophore TAMRA and a quencher BHQ2 are labeled in the stem region of the hairpin probe H1. Therefore, the HCR assembly can draw the fluorophore apart from the quencher and activate an intense fluorescence response. At alkaline pH (Scheme 1c), C+ bases are deprotonated into C, which triggers dissociation of the triplexes of a**-a-a* and b**-bb* in the HCR assembly. The dissociated triplexes decrease thermodynamic stability of the HCR assembly, in which H1 and H2 are spontaneously refolded into the hairpin conformation due to an entropy-favored process, thereby resulting in disassembly of the HCR polymer. The refolded hairpin state of H1 also induces the fluorophore and the quencher into close proximity, resulting in a weak fluorescence signal. Based on this design, a pHresponsive, fully-reversible HCR assembly can be

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achieved, which delivers intense fluorescence signal at acidic pH indicating the assembly process and displays weak fluorescence signal at alkaline pH indicating the disassembly state. To our knowledge, our design has generated the first pH sensor using fully-reversible HCR assembly. Compared to existing pH responsive DNA devices that deliver uniformly distributed fluorescence signals, our design affords HCR assembles with highly localized fluorescence responses. This spatially resolved signal improves the detection sensitivity and affords extra selectivity, since the interferences from fluorescence activation by degraded probes can be discriminated from the spatial distribution of the signals. Therefore, our design can provide a useful sensor to detect intracellular pH with high sensitivity and excellent selectivity.

Figure 1. a) Gel electrophoresis of HCR system. Lane 1, 1 μM H1 and H2; Lane 2, 1 μM I and 1 μM H1; Lane 3, 1 μM H1 and H2 plus 1 μM I; Lane 4, 1 μM H1 and H2 plus 200 nM I; Lane 5, 1 μM H1 and H2 plus 1 μM I at pH 5.0/8.0 followed by reversing pH to 8.0/5.0. b) Fluorescence spectral responses at pH 5.0 for H1 and H2 plus I (red), H1 plus I (orange), H1 plus H2 (cyan), and at pH 8.0 for H1 and H2 plus I (green), H1 plus I (blue), H1 plus H2 (pink). c) Fluorescence intensities of reversibility study between pH 5.0 (I) and 8.0 (II). d) Fluorescence spectral signals obtained at pH 4.5, 5.0, 5.2, 5.5, 5.7, 6.0, 6.2, 6.5, 7.0, 7.5 and 8.0. Insert is plot of fluorescence intensities at 580 nm versus pH values. e) AFM images of reversibility study between pH 8.0 (I, III) and pH 5.0 (II, IV).

We firstly demonstrated the reversible assembly of our HCR system using gel electrophoresis (Fig. 1a). In the absence of initiator I, the mixture of H1 and H2 only showed one band at pH 5.0 or 8.0, which was ascribed to the same mobility of H1 and H2. When the mixture incubated with I at pH 5.0, multiple characteristic ladderlike bands with higher molecular weights were obtained,

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evidencing formation of the HCR assembly. This result implied that formation of the triplex in the toehold region was able to trigger HCR assembly. As anticipated, these bands disappeared when the HCR assembly exposed to pH 8.0 (Lane 5 in left image), indicating disassembly of the HCR polymer at basic pH. Furthermore, the entropy gains were calculated to be 60.4 and 81.1 cal mol-1 K-1, respectively, for the disassembly of I from H1 and H1 from H2.21 In contrast, there was no substantial change in the enthalpy in the disassembly because the base pairs remained unchanged.21 These calculations conformed the entropy-favored mechanism for the disassembly reaction. We also found that incubation of H1 and H2 with or without I at pH 8.0 did not give new bands, revealing that there was no HCR assembly at alkaline pH. In contrast, when HCl was added in the alkaline mixture to adjust the pH to 5.0, typical ladder-like bands for HCR assembly appeared (Lane 5 in right image). These results gave clear evidences for the fully-reversible pH-controlled HCR assembly. The fluorescence spectra further validated the pHresponsible fully-reversible HCR assembly (Figure 1b). The mixture of H1 and H2 at pH 5.0 or 8.0 only gave weak fluorescence peaks, indicating no HCR assembly in the absence of I. Incubating H1 with I at pH 5.0 gave an enhanced (~2.5 fold) fluorescence peak, verifying that formation of the triplex in the toehold region at acidic pH was able to open hairpin H1. Incubation H1 and H2 with I at pH 5.0 generated a further enhanced (>4-fold) fluorescence. This further enhanced fluorescence was attributed to signal amplification of HCR assembly, which opened more than one hairpin H1 with a single initiator. A control experiment of incubating H1 and H2 with I at pH 8.0 did not show appreciable fluorescence enhancement, confirming that the triplex, which did not form at basic pH, was essential for the HCR assembly. A further reversibility study of the HCR assembly was performed by adjusting the mixture of H1, H2 and I between pH 5.0 and 8.0 (Figure 1c). As anticipated, switching of fluorescence signals was fully reversible with all efficiencies over 95%. In addition, the fluorescence intensity of H1 at 580 nm was found to remained unchanged at different pH values (Figure S1 in SI), verifying that the reversible pH responses were dominantly arising from the reversible HCR assembly. AFM images gave further evidences for reversibility of the pH-responsive HCR assembly (Figure 1e). Taken together, the results validated the design for the fully-reversible pH-responsible HCR assembly. Time-dependent measurements of fluorescence intensities at 580 nm during the assembly and disassembly processes revealed that the assembly exhibited a slow kinetics (the reaction completed in ~40 min), while the disassembly displayed a relatively fast reaction (the reaction completed in ~10 min) (Figure S2 in SI). This slower kinetics for HCR assembly was attributed to an increased activation energy derived from highly negative charged backbone of DNA polymers obtained in the assembly, which resulted in strong electrostatic repulsion to monomer H1 or H2, and prevented further extension of the polymers.

We also found that the fluorescence peak gradually decreased with increasing pH from 4.5 to 8.0 (Figure 1d). A dynamic correlation of fluorescence signals to pH values was obtained in the pH range from 4.5 to 7.0, which covered the physiological pH values of living cells. This result indicated that the pH-responsive, fully reversible HCR assembly afforded a useful sensor of pH monitoring in living cells.

Figure 2. Fluorescence images of HCR system for nigericintreated HeLa cells at pH 5.0 (1 and 3) and 8.0 (2 and 4). a) Fluorescence, b) Merge with DIC. Scale bar: 20 μm.

Next, we investigated the potential of the pHresponsive HCR assembly for pH imaging in living cells. HeLa cells were transfected with DNA probes H1, H2 and I using Lipofectamine 3000 for 1.5 h, followed by treatment with 10 μM H+/K+ ionophore nigericin which allowed homogenizing H+ concentrations inside and outside the cells (Figure 2). When the cells incubated at pH 5.0, many highly bright spots were obtained. These spots were ascribed to the HCR assembly with a large number of activated TAMRA fluorophores, evidencing formation of the HCR assembly at acidic pH. In contrast, when the cells were incubated using another pH 8.0 buffer, the bright fluorescence spots disappeared, suggesting that the HCR products became disassembled at basic conditions. Interestingly, re-incubation of the cells with an acidic medium (pH 5.0) recovered highly fluorescent spots in the cells, and re-change to a basic buffer again diminished the fluorescence spots. This finding revealed fully-reversible assembly of the HCR polymer in the cells in response to pH changes. A close interrogation using time-dependent imaging showed that, when the cells exposed to an acidic medium (pH 5.0), the fluorescence signals increased with time and reached a stable output after 40 min; while the cells exposed to pH 8.0, the fluorescence responses became diminished after 10 min (Figure S3 in SI). These time-dependent observations were consistent with those obtained in vitro, implying the realization of the fully-reversible, pHresponsive HCR assembly in living cells. Further experiments were performed for fluorescence imaging of HeLa cells exposed to media with varying pH values (Figure S4 in SI). We found that punctate fluorescence signals gradually decreased with increasing pH, demonstrated the ability of the HCR assembly for quantitative pH imaging in living cells. A further colocalization study gave a Pearson’s coefficient (0.28) for the fluorescence signals and LysoTracker (Figure S5 in SI),

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revealing predominant cytosolic localization for the fluorescence spots. This result suggested efficient escape of DNA probes from lysosomes, and implied the potential of the HCR system for monitoring cytosolic pH variations in living cells.

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Experimental methods including gel electrophoresis analysis, AFM imaging, fluorescence measurements, cell culture and fluorescence imaging as well as additional figures. These materials are available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author [email protected]; [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by NSFC (21527810, 21205034, 21521063).

REFERENCES Figure 3. Images of HCR system in HeLa cells (1) in PBS (pH 7.4), (2) treated with 100 μM H2O2, 3) treated with 100 μM NaClO. a) Fluorescence, b) Merge. Scale bar: 20 μm.

We then explored the utility of the pH-responsive HCR assembly to probe the influence of reactive oxygen species such as H2O2 and NaClO on cytosolic pH in living cells (Figure 3). At pH 7.4, HeLa cells showed moderate fluorescence, indicating that there were HCR assemblies in the cytosol. When the cells treated with 100 μM H2O2, much higher fluorescence signals were obtained. This finding implied a dramatic decrease in intracellular pH, which was ascribed to the generation of acidic substances caused by oxidative stress.22 Surprisingly, after treated with 100 μM NaClO, the cells did not show obvious changes in fluorescence signals, suggesting no remarkable pH change in the cytosol. This finding was consistent with the previous report that the elevated level of ClO− could not elevate intracellular acidic substances.14 These results demonstrated that the pH-responsive HCR assembly afforded a useful approach for visualizing pH fluctuations under varying stimuli in living cells. In conclusion, we have developed a novel pHresponsive, fully-reversible HCR assembly sensor for intracellular pH sensing and imaging. To our knowledge, this is the first fully-reversible HCR assembly system induced by triplex toehold mediated strand displacement. The HCR assembly sensor has shown dynamic responses in physiological pH ranges with excellent reversibility and demonstrated the potential for in vitro detection and livecell imaging of pH. Compared to existing pH-responsive DNA devices that delivers uniformly-distributed fluorescence signals, our design affords HCR assembles with highly localized fluorescence responses. This spatially resolved signal affords advantages of improved sensitivity and better selectivity. Therefore, the HCR assembly sensor can provide a useful approach for pHrelated cell biology study and disease diagnostics.

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