Programming Enzyme-Initiated Autonomous DNAzyme Nanodevices

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Programming Enzyme-Initiated Autonomous DNAzyme Nanodevices in Living Cells Feng Chen, Min Bai, Ke Cao, Yue Zhao, Xiaowen Cao, Jing Wei, Na Wu, Jiang Li, Lihua Wang, Chunhai Fan, and Yongxi Zhao ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06728 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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Programming Enzyme-Initiated Autonomous DNAzyme Nanodevices in Living Cells Feng Chen, ‡a Min Bai, ‡a Ke Cao, a Yue Zhao, a Xiaowen Cao, a Jing Wei, a Na Wu, a Jiang Li, b Lihua Wang, b Chunhai Fan, b and Yongxi Zhao*a

a

Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of

Life Science and Technology, Xi’an Jiaotong University, Xianning West Road, Xi’an, Shaanxi 710049, P. R. China

b

Division of Physical Biology & Bioimaging Center, Shanghai Institute of Applied Physics,

Chinese Academy of Sciences, Shanghai 201800, P. R. China.

* E-mail: [email protected].

DNA computing • nanoassembly • molecular device • enzyme catalysis • genetic circuit

ABSTRACT

Molecular nanodevices are computational assemblers that switch defined states upon external stimulation. However, interfacing artificial nanodevices with natural molecular machineries in living cells remains a great challenge. Here we delineate a generic method for programming

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assembly of enzyme-initiated DNAzyme nanodevices (DzNanos). Two programs including split assembly of two partzymes and toehold-exchange displacement assembly of one intact DNAzyme initiated by telomerase are computed. The intact one obtains higher assembly yield and catalytic performance ascribed to proper conformation folding and active misplaced assembly. By employing MnO2 nanosheets as both DNA carriers and source of Mn2+ as DNAzyme cofactor, we find that this DzNano is properly assembled via a series of conformational states in living cells, and operates autonomously with sustained cleavage activity. Other enzymes can also induce corresponding DzNano assembly with defined programming modules. These DzNanos not only can monitor enzyme catalysis in situ, but also will enable the implementation of cellular stages, behaviors and pathways for basic science, diagnostic and therapeutic applications as genetic circuits.

DNA nanodevices are computational assemblers that switch defined molecular states upon external stimulation. 1-4 They have been motivated by the goal of building cellular monitor systems and smart therapeutic tools. However, interfacing artificial nanodevices with natural molecular machineries in living cells remains a great challenge. Recently, a few cellular DNA nanodevices have been built via simple structure switching or sequence assembly. 2,3,5-11 Most of them are initiated by ions, pH and small molecules, and work as biosensors. Nevertheless, they lack programmed module design for versatile response to active biomacromolecules such as


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enzymes, and can not implement cellular modulation as genetic circuits. Moreover, molecularly crowded and dynamically changing cellular environments still adversely affect the assembly yield and active performance of DNA nanodevices. Especially, multi-stage nanodevices which contain long assembly modules and many interacting components are difficult to construct in living cells. The diffusion efficiency and hybridization rate of DNA modules in the cytoplasm are much poorer than those in well-mixed buffers. 1,10,12 A decrease of difusion efficiency about 5-100 times is demonstrated depending on the size and shape of molecules.12 And external supplement of metal ions into cultured cells was required to activate DNAzyme-based devices. 6,13

This manipulation is tedious and not applicable in practical settings. As macromolecular

biological catalysts, enzymes determine the metabolic processes and are closely involved in diseases. They play more important and versatile roles than ions and small molecules. Yet cellular enzyme-initiated assembly and active operation of DNA nanodevices is rarely explored. Therefore, programming enzyme-initiated DNA nanodevices and executing designated tasks in living cells with similar efficacy as in vitro remain necessary and challenging.

In this work, we delineated a generic method for programming assembly of enzymeinitiated DNAzyme nanodevices (DzNanos). Telomerase is used as a model initiating enzyme. Both split assembly program of two partzymes and toehold-exchange displacement assembly program of one intact DNAzyme are computed. MnO2 nanosheets were employed to facilitate cellular uptake of DNA probes, and as generators of DNAzyme cofactor Mn2+ for the assembly


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and active operation of DzNanos in living cells. We find that the intact DzNano obtains higher yield and catalytic cleavage ability ascribed to proper conformation folding and active misplaced assembly. It not only can monitor enzyme catalysis, but also will enable the implementation of cellular modulation for diagnostic and therapeutic applications.

RESULTS AND DISCUSSION

Scheme 1 depicts the design concepts. Each assembly program has three DNA modules: telomerase recognition (TR), DNAzyme bridging (DB) and DNAzyme substrate (DS). The DS modules of two programs are the same one (fluorescent probes or cellular RNAs). In the intact assembly program, the DB contains intact DNAzyme (middle catalytic core and two flanking

Scheme 1. Design and programming of telomerase-initiated assembly of DzNanos operating in living cells. TR, DB and DS indicate telomerase recognition, DNAzyme bridging and DNAzyme substrate modules, respectively. The extended TR module can successively assemble DB module


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and DS module into active Dznanos. DzNanos would catalyze the hydrolyzation of fluorescent DS modules or cellular RNAs for telomerase monitoring or gene modulation.

substrate arms) caged in a stem-loop structure, and its 5' end overhangs as a toehold (complementary with telomeric sequences). It can connect DS module and extended TR module, which is inspired by bridging two signal amplification modules in our previous work.14 Corresponding TR module is designed as a chimeric DNA probe with telomerase extension (TE) primer at 3' end and displacement sequence at 5' end. The displacement sequence is complementary with partial sequence of the DB module in the stem part, yet it is disabled to trigger the strand displacement reaction without telomeric sequence (AGGGTT). In the presence of telomerase, TE primer in TR module is extended with repetitive telomeric sequences at 3' end, namely telomerase extended TR (teTR). It can rapidly form stem-loop structure via intramolecular hybridization as stem-loop TR (slTR), flanking two terminal overhangs. These two overhangs are tightly pulled. They can trigger toehold exchange-mediated strand displacement with DB module, forming slTR/DR duplex. As a result, two substrate arms in DB module are released for the binding to DS module. In this way, the intact DzNano (slTR/DR/DS triplex) is assembled. Notably, Mn2+ is generated for the folding and catalysis of DzNano as the DNAzyme cofactor. This DzNano is capable of cleaving many molecules of the substrate probe under multiple turnover conditions. If the substrate probe is labelled with a fluorophore and a matched quencher on each side of the cleavage site, amplified fluorescence signal will be


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provided for telomerase activity monitoring. Using cellular RNAs as DNAzyme substrates, telomerase-triggered modulation of gene expression could also be allowed. This assembly mechanism can be expressed as the following reactions [Equations (1), (2) and (3)]: kA

teTR → slTR

(1)

kB

slTR + DB → slTR/DR

(2)

kC

slTR/DR + DS → slTR/DR/DS

(3)

The net reaction of the system [Equation (4)]

teTR + DB + DS → slTR/DR/DS

(4)

A long stem in slTR increases kA, and contributes high thermostability and slTR yield. Based on the nature of toehold exchange reaction,15-17 kB increases as the decrease of the length of unexposed toehold in the stem of DB module. However, short unexposed toehold allow unextended TR module destroy the stem-loop structure of DB module, leading to non-specific assembly. In addition, a tradeoff between the binding affinity to DS and release rate of cleaved substrates is also required to obtain moderate kC for high turnover number.

For split assembly program, the TR module and DB module each contains one arm complementary with DS module, partial catalytic core and 6 complementary bases (spacer) close to catalytic core. Besides, TR module is designed with TE primer at 3' part for telomerase


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recognition, and DB module has telomeric complementary sequences at 5' end. All of these complementary parts cannot form thermostable duplexes at reaction temperature (37 oC) due to low melting temperatures (~ 25 oC). Thus, these dissociative modules are disabled to initiate DzNano in the absence of telomerase. In contrast, telomerase can recognize and extend TE primer in TR module to produce repetitive AGGGTT sequences, leading to its intramolecular hybridization. Then, this stem-loop TR module steadily interacts with DB module at its 5' and spacer parts, assembling partzymes into a full DNAzyme with an active catalytic core. After the binding of DS module, split DzNano is properly assembled. This assembly program is inspired by three-way DNA junction structure reported in our previous works. 18-20 And its mechanism can be expressed as similar equations to those of intact assembly program except with different DNA structures and reaction constants. Notably, the constant of reaction between stem-loop TR module and DB module, k'B, significantly affects both assembly yield and catalytic activity of split DzNano.

We firstly investigated the assembly of DzNanos by fluorescence melting curve, gel electrophoresis and atomic force microscopy (AFM). As shown in Figure S1, melting peaks of high temperatures were observed in each curve of extended TR modules rather than TR modules, indicating the generation of thermostable stem-loop DNA at low temperatures. The samples of partial assembly (extended TR module/DB module) and full assembly (extended TR module/DB module/DS module) also induced similar melting peaks to those of extended TR modules. They


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couldn’t exactly confirm the assembly of DzNanos. In the electrophoretogram of Figure 1A, prominent bands were exhibited in lane 1-3 and 1’-3’, indicating the formation of DNA stem or duplexes. Their mobility apparently increased along with the number, suggesting the ordinal construction of DzNanos. For split DzNano, abundant unassembled TR modules and remarkable byproduct were each observed in Lane 2 and Lane 1, causing its lower yield than that of intact one (Lane 1’). The byproducts may be DNA duplexes of extended TR module/DS module or DB module/DS module. It indicates that the value of kB is higher than that of k'B. Finally, AFM is

Figure 1. In vitro characterization of programmable assembly of DzNanos. (A) Gel electrophoresis analysis. Lane 4-1 (split assembly) and 4’-1’ (intact assembly) each indicate TR


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module only, extended TR module only, extended TR module + DB module, and extended TR module + DB module + DS module. (B) AFM characterization of the size and morphology of split DzNano (left) and intact DzNano (right).

used to directly characterize the size (about 10 nm) and morphology of DzNanos (Figure 1B). Furthermore, theoretical prediction of DzNanos was also performed by Nucleic Acid Package (NUPACK) software. The stem-loop secondary structures of extended TR modules under related reaction conditions were showed in Figure S2. Figure S3 depicted the lowest free energy structures of these two DzNanos, which hold the structures as we confirmed.

After the verification of DzNano conformations, their catalytic performance were evaluated by using fluorescent probes as DS modules. Telomerase-induced signals are observed in both DzNanos, and the catalytic efficiency of intact one is much higher than that of split one (Figure S4). Notably, the kB values in Equations (2), indicating the assembly of slTR and DB into slTR/DR duplex, effect the yields of both telomerase-specific and non-specific assembly of intact DzNano. As presented in Figure 2A, increased fluorescence signals and background intensity were observed with the decrease of unexposed toehold length as discussed above. Figure 2B indicated the yield of intact DzNano under varied concentrations of extended TR module and DB module. It supports that this assembly mechanism functions as designed: the simultaneous consumption of these DNA modules results in the production of stoichiometric DzNano. In addition, the effects of concentrations of TR module and DS module on intact


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DzNano assembly were evaluated as depicted in Figure 2C and 2D. In split DzNano, the k'B constant was investigated using four different DB modules (Figure S5). We found that its catalytic activity was much lower than that of intact DzNano. Misplaced assembly of DB module to extended TR module in split DzNano may cause the low assembly yield and poor catalytic performance (Figure S6A). In intact assembly program, misplaced binding between extended TR module and DB module may also exist (Figure S6B); however, the strand displacement process still occurs based on a remote toehold mechanism. 21

Figure 2. Catalytic performance of intact DzNano using fluorescent probes as DS modules. (A) Toehold exchange-mediated assembly with different unexposed toehold length. (B) The


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assembly yield of DzNano under varied concentrations of extended TR module and DB module. The effect of concentrations of TR module (C) and DS module (D) on intact DNAzyme nanodevice.

The metal ion cofactors are indispensable for the conformation folding and catalysis of DNAzyme. The catalytic core sequence can form a specific binding pocket for these cofactors. As we expected, the intact DzNano operates reliably in the presence of as low as 0.1 mM Mn2+, whereas intracellular level Mg2+ (about 0.5 mM) led to unconspicuous signal increase (Figure 3A). Therefore, supplying DNAzyme cofactors is necessary to enable catalytic performance in

Figure 3. (A) Divalent metal ion-dependent catalytic performance of DzNano. The assembly and cleavage reactions were carried out in M2+-free buffer (10 mM Tris-HCl, 50 mM NaCl, 100 mM


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KCl, pH 7.5) with Mg2+ or Mn2+ of different concentrations at 37 oC for 60 min. (B) Characterization of MnO2 nanosheets by TEM. (C) AFM analysis of MnO2 nanosheet. (D) GSHinduced reduction of MnO2 nanosheets to Mn2+ as DNAzyme cofactor.

cellular environments. MnO2 nanosheet was used in this work due to its strong DNA adsorption capacity and fast reduction to Mn2+ in living cells. Compared to MnO2 nanoparticles, nanosheets are rapidly reduced in cellular environments. Considering Zn2+ and Mg2+ also as representative DNAzyme cofactors, biodegradable ZnO and Mg/Al nanomaterials may also useable. The characterization was shown in Figure 3B by transmission electron microscopy (TEM). Figure 3C presents ~2 nm topographic heights of MnO2 nanosheets by AFM. Cellular reduction of MnO2 nanosheet was simulated in vitro by using glutathione (GSH, intracellular level of ~2 mM) as a model reductant (Figure 3D). Moreover, the excellent biocompatibility of MnO2 nanosheets (up to 100 µg/mL) was confirmed (Figure S7). This nanosheet concentration is approximately equal to 1.2 mM Mn2+, allowing cellular generation of enough Mn2+ (0.1 mM) for intact DzNano.

Finally, cellular assembly of telomerase-specific DzNanos and corresponding operation in living cells were investigated. As depicted in Figure 4A and S8, strong signals were observed in two tumor cell lines (MCF-7 and HeLa) incubated with intact DzNano using telomerase-specific TR module. In contrast, the control nanosystem using TE primer-deleted TR module disables the assembly of DzNano, which exhibits very low fluorescence signal approximately equal to background. This negative TR has random sequence rather than poly (T) at the 3' end, avoiding


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the binding to abundant poly (A) sequences in mRNAs and lncRNAs. Normal cell lines HEK 293T showed dim fluorescence, indicating up-regulation telomerase activities in tumor cells. Another negative control using standard TE primer instead of TR module also failed to form an


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Figure 4. (A) Microscope images of cells treated with positive and negative DzNanos. The intensity information (0-256) for each pixel was extracted by MATLAB. [1] and [0] indicates the successful and failing assembly of intact DzNano, respectively. (B) Fluorescence intensity distributions in (A).

active DzNano (Figure S9A). In addition, non-MnO2 assembly of DzNano and corresponding operation in MCF-7 cells were limited with low efficiency (Figure S10). These results demonstrated cellular telomerase-specific programmable assembly of intact DzNano and its excellent catalytic performance in tumor cells.

The intensity information for each pixel of cell images in Figure 4A was extracted by MATLAB. Corresponding scatter plot showing intensity distribution confirmed the intracellular performance of our intact DzNano (Figure 4B). And at least 60-fold fluorescence enhancement for cancer cells was obtained compared to the negative control with TE primer-deleted TR module (Figure S8C). As we known, the sensitivity is comparable with those of previously reported works. 22-27 On the other hand, the operation of MnO2 nanosheet-integrated split DzNano in living cells was also evaluated. As depicted in Figure S9B, no remarkable fluorescence signals were observed in tumor cell lines.

Overall, intact DzNano outperformed split one in both well-mixed buffers and densely packed cellular environments. Though assembling split partzymes into full DNAzymes in living cells was reported for gene regulation and imaging analysis 8,28,29, they only exhibited residual


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catalytic activity. Moreover, intact DNAzyme also showed low catalytic efficiency in cellular conditions due to improper conformation folding and limited binding affinity of DNAzyme to substrate. 13,30-33 External supplement of high concentrations of metal ions into cultured cells was required to activate those DNAzymes. 6 This treatment is tedious and impractical for clinical application. Notably, other enzymes such as DNA repair proteins can also initiate corresponding DzNano assembly by programming defined modules. Our next work will focus on the implementation of cellular state machines and gene modulation as genetic circuits. Multienzymeinitiated or multi-functional DzNanos may also by fabricated by integrating different sets of DNA modules. They can execute multiple tasks, and are useful for the monitoring and regulation of cell pathways or networks.

CONCLUSIONS

In summary, we have built cellular telomerase-initiated DzNanos via programmable DNA assembly. Split and intact assembly programs were designed. The intact DzNano presented much higher yield and catalytic activity. MnO2 nanosheets were used as DNA carriers to facilitate cellular uptake of DNA probes, and as DNAzyme cofactor generators for cellular assembly and folding of DzNanos. The intact DzNano operated autonomously in living cells with high cleavage capacity. At least 60-fold enhanced signal was obtained for the monitoring of telomerase catalysis in cancer cells. Other enzymes can also induce corresponding DzNano assembly with defined DNA modules. These DzNanos not only can monitor enzyme catalysis in


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situ, but also will enable the implementation of cellular modulation, behaviors and pathways for diagnostic and therapeutic applications as genetic circuits.

METHODS

Materials. Tetramethylammonium hydroxide pentahydrate, hydrogen peroxide (H2O2, 30 wt%), manganese chloride tetra-hydrate (MnCl2·4H2O), L-glutathione reduced (GSH), were purchased from Sigma-Aldrich (St Louis, MO, USA). All the chemicals were of analytical grade and used as received without further purification. The Milli-Q water (resistance>18.2 MΩ) used for solution preparation and reaction was RNase-free. The oligonucleotides used in this work (Table S1) were synthesized by Sangon Biological Co. Ltd (Shanghai, China) except that Substrate probe was obtained from TaKaRa Biotechnology Co. Ltd. (Dalian, China). DNA marker and RNase inhibitor were also obtained from TaKaRa Biotechnology Co. Ltd.

Preparation of telomerase-initiated DzNanos. In a typical experiment for the assembly of intact DzNano, the reaction is performed in 10 µL of annealing buffer containing 75 nM TR module (or Extended TR modul), 100 nM DB module and 600 nM Substrate probe. The mixture is incubated at 37 oC for 60 min in LightCycler 96 for real-time fluorescence monitoring. For the assembly of split DzNano, the conditions are similar to those of intact DzNano except using its own TR modul and DB module (Table S1).


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Characterization of DzNanos by AFM. AFM tips (model SNL-10; Veeco-Digital Instruments) are used in this work. A freshly cleaved mica surface for sample mounting is firstly prepared. 0.5% (wt/vol) APTES solution is added to the center of the mica surface for 2 min. After rinsing the mica surface vigorously with Milli-Q water followed by its dry with nitrogen gas, 20 µl solution of DNAzyme nanodevice is pipetted onto the center of the mica surface for incubation of 15 min. Then, 80 µl of annealing buffer is added. The image is operated in PeakForce tapping mode under solution using super-sharp tips (model SNL-10) with a spring constant of 0.35 at a scanning rate of 1 Hz.

Melting curve and gel electrophoresis analysis. LightCycler 96 (Roche Applied Science, Mannheim, Germany) was used to record real-time fluorescence signals and melting curves. 400 nM DNA modules and 0.5 x SYBR Green I were mixed in 10 µL of annealing buffer (20 mM Tris-HCl, 10 mM NaCl, 100 mM KCl, 10 mM MgCl2, pH 7.5). After incubation of 10 min at 37 o

C, the melting curves of the mixtures are recorded. Then, products were analyzed by 3.5%

agarose gel electrophoresis in 1× TBE buffer at a 70 V constant voltage for 60 min at room temperature. The gel was visualized by Syngene G:BOX Imaging System.

Synthesis and characterization of MnO2 nanosheets. Manganese dioxide nanosheets were synthesized according to previous reports.30 Briefly, a mixed aqueous solution (20 mL) containing 3 wt % H2O2 and 0.6 M tetramethylammonium hydroxide was added to 0.3 M MnCl2 (10 mL) solution quickly within 15 s under stirring. The dark brown solution was stirred


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vigorously overnight in the open air at room temperature. Subsequently, the bulk manganese dioxide were collected via centrifugation and then washed with water and methanol. This purification cycle was repeated at least for twice. And then the as-prepared bulk MnO2 was dried at 60 oC. To receive the MnO2 nanosheets, 10 mg bulk MnO2 was dispersed in 20 mL water under ultrasonication (>10 h). The shape and size of the MnO2 nanosheets were examined via the HT7700 transmission electron microscope (Hitachi, Japan) with a tungsten filament at an accelerating voltage of 100 kV.

Cellular toxicity assay of MnO2 nanosheets. The 3-(4, 5-Dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used to evaluated cell viability. MCF-7 cells were seeded in a 96-well microplate with 40,000 cells/well and 5 wells for each concentration. The cells were incubated at 37 oC for 12 h. Subsequently, cells were treated with MnO2 nanosheets of different concentrations (0-100 µg/mL) at 37 oC for 24 h. The medium was removed, and 10 µL of sterilefiltered MTT stock solution in PBS (4.0 mg/mL) was added to each well. After 30 min incubation, the absorbance measurement at 490 nm was carried out by using a microplate reader (Tecan Infinite® F50).

Cell culture and imaging analysis. Mammalian cell lines (HeLa, MCF-7 and HEK 293T) were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% of heat-inactivated fetal bovine serum and 1% antibiotics penicillin/streptomycin (100 U/mL) in a humidified incubator containing CO2 (5%) at 37 oC. Tumor cell lines (HeLa and MCF-7) and normal cell lines HEK


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293T were seeded in 8-well Chambered Cover Glass (Cellvis, USA) at 60,000 cells per well at 37 oC for overnight, then incubated with 200 µL of serum-free culture media containing 20 µg/mL MnO2/DNA modules composite (containing 100 nM TR module, 100 nM DB module, and 400 nM Substrate probe) for 8 h. The cells were washed three times with PBS before imaging. All fluorescence images were acquired using Nikon A1 laser scanning confocal microscopy. A 488 nm laser was used as the excitation source. Fluorescence enhancement factor was defined as the ratio of mean fluorescence intensity of cells. The mean fluorescence intensity of cells is determined by averaging the fluorescence intensity of randomly selected cells measured by ImageJ software after subtracting the background.

Data extraction by MATLAB. The fluorescence images of cells were opened by MATLAB. And a serial of commands were added in the command line window to convert the cell image into two-dimensional matrixes that contain the intensity information (0-256) of three color channels (R: red, G: green, and B: blue). The matrix of green channel (FAM fluorescence) was used to depict the intensity distribution.

ASSOCIATED CONTENT

Supporting Information. A list of all oligonucleotide sequences, and supporting figures are provided.

The authors declare no competing financial interest.


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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].

Author Contributions ‡

These authors contributed equally.

ACKNOWLEDGMENT This research was financially supported by the National Science Foundation of China (No. 21475102 and No. 31671013), the China Postdoctoral Science Foundation (No. 2017M613102), the Fundamental Research Funds for the Central Universities (xjj2017039) and “Young Talent Support Plan” of Xi’an Jiaotong University.

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Programming assembly of enzyme-initiated autonomous DNAzyme nanodevices in living cells was reported to monitor enzyme catalysis and implement gene modulation.


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Programming Enzyme-Initiated Autonomous DNAzyme Nanodevices

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