Light-Mediated Spatiotemporally Dynamic Assembly of DNA Nanostructures in Living Cells Regulates Autophagy

Introduction

The assembly of biomolecules in living cells is an important way to regulate cell activities.^1–5 The formation of intracellular artificial assemblies can also lead to a series of biological effects, such as interference with cell behavior and intervention in disease progression.^6–9 In recent years, some intracellular artificial assembly systems have been developed to study their mechanisms and biological effects, during the interactions between artificial assemblies and subcellular structures.^10–13 For example, exogenous synthetic hydrogels and RNA particles were used to reconstruct the nucleation process of biological functional entities.¹⁴ Artificial organelles with biological functions were constructed via intracellular in-situ assembly of proteins.⁶ The free radical polymerization of unnatural polymers in a complex living-cell environment was realized and used to regulate cell functions and behaviors.¹⁵ These studies on intracellular artificial assembly hugely depend on precise and controllable systems of materials chemistry.

As a biomacromolecule, deoxyribonucleic acid (DNA) can be used as such a precise and controllable materials chemical system, which enables the construction of diverse functional DNA modules, presenting molecular recognition, stimulus responsiveness, and biological activities.¹⁶ In response to environmental stimuli such as pH,¹⁷ enzymes, and ions,^18,19 functional DNA modules can dynamically assemble with each other, forming higher-level architectures.²⁰ In particular, a few DNA-based materials chemistry systems have been constructed to realize in-situ assembly in living cells.¹⁰ Almost all of these established assembly systems passively respond to cellular endogenous stimuli. The introduction of exogenously controlled modules to further enhance the controllability of assembly system is a promising route.

In this work, we introduced a photocleavable chemical bond and an acidity-responsive i-motif moiety in DNA molecules and therefore realized the assembly of DNA nanostructures by both an exogenously introduced trigger and cellular endogenous stimuli. The assembly was implemented under the dual-mode control of ultraviolet (UV) light and acidic conditions through a logical AND-gate. A DNA nanostructure composed of i-motif moiety-containing branched DNA, photocleavable (PC) bond-containing linker, and tumor cell-targeting aptamer was constructed. By introducing UV light at different endosomal/lysosomal stages to cancer cells, the disassembly and reassembly of DNA nanostructures occurred, synergistically mediated by the lysosomal acidity microenvironment. Based on the light-mediated spatiotemporally dynamic assembly of DNA nanostructures in living cells, the regulation of autophagy and related cell behaviors was investigated.

We designed two kinds of Y-shaped branched DNA, YA and YB, as the basic building-blocks of this system, namely DNA composed of three-arm double-stranded DNA elongating from a branched point with three cytosine-rich single-stranded sticky ends. The branched DNA were synthesized from three single-stranded DNA (ssDNA) with partially complementary sequences ( Supporting Information Table S1). By introducing a PC bond-containing linker, the branched DNA were assembled with each other and formed DNA nanoparticles (DNPs) with the assistance of AS1411 aptamers as sticky-end blockers. To effectively block the sticky ends in DNPs and respond to the acidic microenvironment at the physiological temperature of 37 °C, linker and aptamer extended 18 bases at the 5′ end, and the unwinding temperature between linker and complementary DNA strand were calculated to be 62.4 and 58.8 °C. When DNPs were exposed to UV light, PC linker was photocleaved to produce two nonnucleoside molecules, a spacer oligo, a 5′-phosphorylated oligo, and a 3′-phosphorylated oligo (Scheme 1a). At pH 6.5, since the calculated maximum unwinding temperature between the oligo at the 5′ end of PC linker and the complementary fragment was 35.3 °C, the two fragments dehybridized at 37 °C; as a result, DNPs disassembled into Y-shaped monomer A (YA), Y-shaped monomer B (YB), and fragments. On the other hand, upon pH 5.0, YA and YB reassembled through the hydrogen bonding of i-motif sequences driven by H⁺, forming DNA assemblies (DABs).

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According to our design, DNPs were internalized by cells through the lysosomal pathway.^21,22 DNPs underwent different endosomal/lysosomal stages after cellular uptake. Endosomes and lysosomes contain various nucleases that can degrade DNA molecules, especially ssDNA.^23,24 Under the control of both UV light and the late endosomal/lysosomal acidic environment, disassembly/reassembly were expected to occur via two different routes, called Route 1 and Route 2 (Scheme 1b). Route 1: UV irradiation was introduced at the early endosomal stage, and DNPs disassembled into free branched DNA. Affected by nucleases in the endosome/lysosome, partial i-motif-containing sticky ends (ssDNA) of YA and YB were hydrolyzed.²⁵ In the late endosomal/lysosomal acidic environment, the residual i-motif-containing sticky ends cross-linked, and thus part of the branched DNA formed small assemblies. Route 2: UV irradiation was introduced at the late endosomal/lysosomal stage. The disassembled branched DNA reassembled rapidly triggered by lysosomal H⁺, during which the sticky ends cross-linked through the i-motif,^26,27 and large DABs were formed. We hypothesized that the intracellular assembly process influences autophagy of cells and further affects the distribution of actin.

Experimental Methods

Synthesis of Y-DNA

Three partially complementary ssDNA with equal molar ratios were added to an Eppendorf (EP) tube to synthesize Y-DNA using the specific heating and annealing program, and the buffer was 2-morpholinoethanesulphonic acid (MES) buffer (50 mM MES, 50 mM NaCl, pH 7.0). The program was as follows: heating at 95 °C for 2 min to denature; after holding at 65 °C for 2 min and at 60 °C for 5 min; annealing from 60 to 20 °C at a decreasing rate of 1 °C per 30 s, for 40 cycles; holding at 20 °C for 30 s, and stop at 10 °C.

Synthesis of DNPs

Stoichiometric quantities of the ssDNA for the YA, YB, DNA linker (LK), and DNA aptamer (Apt) were separately added to four EP tubes with a MES buffer (50 mM MES, 50 mM NaCl, pH 6.5). For the preparation of DNPs, in a typical experiment, a stock solution of the building units was prepared in which stoichiometric amounts of YA, YB, LK, and Apt were added to the MES buffer. The stock solutions of branched DNA (20 μM) for synthesis of DNPs were first prepared with an MES buffer (50 mM MES, 50 mM NaCl, pH 6.5). The molar ratio of YA to YB was equal, the molar ratio of YA to linker was 2:3, and that of Apt to YA was 2:1. After 4 μM of YA and 4 μM of YB were mixed with 6 μM of LK and 8 μM of Apt, the mixture was incubated in a shaker (37 °C, 450 rpm) for 6 h to form DNPs.

The topological transformation of DNPs to aggregates mediated by acid and UV light

The synthesized DNPs were concentrated to 1 μg/μL via ultrafiltration, dispersed in the acidic MES buffer (50 mM, 50 mM NaCl, pH 5.0), and then irradiated with UV light (365 nm, 5 W, 2 min) at 10 cm from tube. DNA aggregates were formed.

Investigation of endocytosis pathway

Michigan Cancer Foundation-7 (MCF-7) cells were seeded into confocal dishes (1 × 105 cells per well) and cultured for 12 h. Then the cells were preincubated with several inhibitors that were specific for different endocytosis pathways for 30 min at 37 °C, including chlorpromazine hydrochloride (10 μg/mL) for clathrin-mediated endocytosis; genistein (200 μg/mL) for pit-mediated endocytosis; Me-β-CD (500 μM) for lipid-raft-mediated endocytosis; amiloride (2 mM) for macro-pinocytosis-mediated endocytosis; nystatin (1 mM) for caveolin-mediated endocytosis; and wortmannin (400 nM) for macropinocytosis. Afterwards, the cells pretreated with the inhibitors were incubated with tetramethylrhodamine (TAMRA)-labeled DNPs (20 ng/μL) for 6 h at 37 °C. In addition, the cells untreated with inhibitors were incubated with TAMRA-labeled DNPs for 6 h at 4 °C and used for the control experiment,. Finally, the cells were fixed with 4% paraformaldehyde for 20 min, washed twice with phosphate-buffered saline (PBS), and stained by 4′,6-diamidino-2-phenylindole (DAPI; 5 μg/mL) for another 15 min followed by washing with PBS twice. Finally, the samples were imaged by confocal laser scanning microscopy (CLSM 800 with Airyscan, Zeiss, Oberkochen, Germany).

Colocalization analysis by CLSM

For the cellular uptake, MCF-7 cells were seeded into cell culture dishes and cultured overnight to allow the cells to attach onto the glass bottom. Afterwards, TAMRA-labeled DNPs were added to wells with a final concentration of 20 ng/μL. After incubation for 0, 2, and 4 h, the cells were stained by LysoTracker Green for 2 h and washed three times by PBS. After incubation with Hoechst 33342 (5 μg/mL) for another 15 min, cells were washed three times by PBS. Finally, fluorescence images of cells were obtained by CLSM equipped with a Plan-Apochromat 40× oil immersion objective (NA 1.3).

Lysosomal acidity measurement

Confocal laser scanning microscopy (CLSM) was used to determine lysosomal acidity. MCF-7 cells (1× 10⁵ cells/dish) were seeded into confocal dishes and cultured for 12 h. TAMRA-labeled DNPs were added with a final concentration of 20 ng/μL for 6 h incubation. According to the instructions, lysosomes and nucleus were stained with LysoSensor Green for 2 h and Hoechst 33342 for 15 min, respectively. After staining, cells were washed three times with PBS. MCF-7 cells were imaged by CLSM, and the fluorescent intensity of LysoSensor Green was analyzed by ImageJ.

Transmission electron microscopy of MCF-7 cells slices

For the analysis of changes in intracellular structure, MCF-7 cells were first treated by UV, DNPs, DNP-UV2, and DNP-UV4 for 6 h, respectively. Cells were washed three times with PBS, harvested, and then centrifuged at 100 × g for 5 min. After removing the supernatant, 1.5 mL pure bovine serum was added to suspend cells, and once again centrifuged at 100 × g for 5 min. Next, supernatant was removed, and 1.5 mL 2.5% glutaraldehyde was added slowly to infiltrate cells, followed by 12 h storage at 4 °C. Next, the cells were further fixed with 1 mL 1% osmium tetroxide. The samples were then dehydrated with ethanol and infiltrated by pure resin. The infiltrated samples were placed in a mold and polymerized at 60 °C for 24 h and then cut into 70 nm ultrathin sections using an ultramicrotome (Leica, Wetzlar, Germany; EMUC7). The ultrathin sections were placed on copper grids and stained with uranyl acetate (2% in ethanol) and lead citrate. Transmission electron microscopy (TEM) images of MCF-7 cells were observed using a transmission electron microscope (JEM-1400Flash, Tokyo, Japan).

Detection of autophagy by Ad-mcherry-GFP-LC3B transfection

MCF-7 cells (5×104 cells per well) were seeded into confocal dishes and cultured overnight to allow the cells to attach onto the glass bottom. According to the instruction of manufacturer, 20 μL of Ad-GFP-mcherry-LC3B was added into cells and incubated with cells for 24 h to transfect cells. After that, the supernatant was replaced with fresh culture medium. DNPs were added with a final concentration of 20 ng/μL and incubated with the cells for 6, 8, 10, 12, 24, and 48 h, respectively. At 4 h, cells were irradiated with UV for 2 min. Cells were washed three times by PBS and observed by CLSM.

F-actin staining and measurement of actin filament orientation

MCF-7 cells were seeded into cell culture dishes and cultured overnight to allow the cells to attach onto the glass bottom. DNPs were added into the cells with a final concentration of 20 ng/μL. When DNPs were added for 2 and 4 h, respectively, the cells were irradiated with UV light (365 nm, 5 W) for 2 min. After 6 h incubation at 37 °C, cells were washed three times with PBS, followed by fixation and permeabilization. F-actin was stained with fluorescein isothiocyanate (FITC)-phalloidin. Images of labeled cells were collected with CLSM and analyzed using ImageJ with the Orientation J plugin.

Cell scratch healing assay

MCF-7 cells were seeded into a 6-well cell culture plate and cultured for 12 h. Afterwards straight lines were scratched across the surface of the wells with a p200 pipette tip. Then wells were gently washed twice with medium to remove the detached cells and replenished with fresh serum-free medium. A bright-field microscope (Nikon, Tokyo, Japan) was used to image the initial scratch. And then DNPs were added to wells with a concentration of 20 ng/μL and treated under different conditions. The control group was treated with PBS. The images of the scratch were collected after incubation with DNPs for 24, 48, 72, and 96 h. The areas of scratch were measured with ImageJ software.

Cell viability

The cytotoxicity of MCF-7 cells treated under different conditions was performed by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in a 96-well plate with a density of 1×104 cells per well and incubated for 12 h. Then the cells were incubated with DNPs, DNP-UV2, and DNP-UV4 for 24 h, respectively. The concentration of DNPs was of 20 ng/μL. After 24 h incubation, all groups were washed three times with PBS, and 100 μL MTT containing fresh medium (1 mg/mL) was added into each well. After 4 h incubation, the supernatant was removed gently, and 100 μL dimethyl sulfoxide was added, incubating for another 10 min. The absorbance of the solution at 490 nm was measured by a microplate reader.

Results and Discussion

Assembly of logic gated DNPs

The dynamic disassembly and reassembly of DNP was logically controlled by UV light and acidity. UV light and acidity acted as inputs and passed through the AND-gate consisting of DNPs. The outputs presented ON or OFF states, corresponding to four input signals, (0, 0), (1, 0), (0, 1), and (1, 1). Only in the presence of both inputs can DNPs undergo sequential disassembly and reassembly, leading to structural transition from nanoparticles to assemblies (Figure 1a). Branched DNA with i-motif terminals (YA and YB) were designed and synthesized by programmed annealing, which was verified by 12% native polyacrylamide gel electrophoresis (PAGE). At pH 5.0, YA and YB assembled through the hydrogen bonding of i-motif sequences driven by H⁺ ( Supporting Information Figures S1 and S2). The photocleavage ability of PC linker was tested under irradiation of 365 nm UV light for varied time durations. After UV irradiation for 2 min, the PC linker was completely cleaved ( Supporting Information Figure S3a). The successful synthesis and UV light-triggered disassembly of DNPs in 2 min were further verified by 12% native PAGE (Figure 1b and Supporting Information Figure S3b). The assembly from branched DNA to DABs at pH 5.0 was completed in 1.5 min ( Supporting Information Figure S4). The results of dynamic light scattering show that the initial hydrodynamic diameter of YA was ∼14 nm and increased to ∼256 nm after assembling with YB, linker and aptamer (Figure 1c). We further investigated the influence of the ratio of branched DNA and aptamer on the size of the obtained DNPs. The molar ratio of YA to YB was equal, and the ratio of branched DNA to linker was determined to be 2:3. The ratios of branched DNA to aptamer were set as 1:1, 1:1.5, and 1:2, respectively. With the increase of the proportion of aptamer, the size of DNPs decreased, and the distribution became uniform, indicating that the aptamer as blocker can regulate particle size and distribution. The formation of stable i-motif structure was further confirmed by circular dichroism (CD) spectral analysis. When the buffer of DNPs was adjusted from pH 6.5 to 5.0, the positive peak showed negligible shift (Figure 1d); when UV irradiation was introduced simultaneously, the positive peak at 274 nm redshifted to 285 nm, indicating the formation of i-motif structure (Figure 1e).²⁸ As a control, DNPs without PC modification were tested, which showed negligible peak shift at pH 5.0 with UV irradiation ( Supporting Information Figure S5). From TEM images, DNPs showed spherical morphology and an average diameter of 217 nm at pH 6.5 (Figure 1f). Upon 2-min UV irradiation, DNPs disassembled into uniformly distributed small particles (Figure 1g). Next, the pH of the solution was adjusted from 6.5 to 5.0 to study the reassembly of DNPs. DNPs without UV irradiation showed spherical morphology without change of size (Figure 1h). In contrast, after UV irradiation, porous network microstructure formed, characterized by TEM, atomic force microscopy, and scanning electron microscopy (Figure 1i and Supporting Information Figure S6), indicating that branched DNA reassembled through the cross-linking of terminal i-motif sequences in sticky ends.

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The real-time localization of DNPs in cells during internalization

The process of cellular internalization toward DNPs was investigated. The results of flow cytometry showed that the uptake efficiency of DNPs by MCF-7 cells gradually increased with the extension of incubation time in 6 h ( Supporting Information Figure S7). To determine the cellular uptake pathway, MCF-7 cells were treated with specific endocytosis inhibitors. and the internalization of DNPs was studied by CLSM. Compared with the control group without inhibitors, cellular uptake was inhibited by chlorpromazine (clathrin-mediated endocytosis inhibitor) and 4 °C hypothermia (Figure 2a and Supporting Information Figure S8), reflecting the fact that the cellular uptake was clathrin-mediated and energy-dependent; in this process, nanoparticles were first internalized by early endosomes and then translocated to late endosomes/lysosomes.²⁹

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To visualize the real-time localization of DNPs in cells during internalization, early endosomes and DNPs were labeled with CellLight probe and TAMRA, respectively, and observed by CLSM. After incubation with MCF-7 cells for 2 h, the fluorescence signal of TAMRA (red) overlapped with CellLight (green), indicating that DNPs colocalized with early endosomes (Figure 2b,c and Supporting Information Figure S9). When the incubation time was extended to 3 and 4 h, the fluorescence signal of TAMRA partially separated from CellLight, indicating that most of the DNPs left from early endosomes (Figure 2d and Supporting Information Figure S9). DNPs translocated to late endosomes/lysosomes after 4 h incubation, which was observed via the luminescence colocalization of DNPs and LysoTracker lysosome probe (Figure 2e–g and Supporting Information Figure S10). The Pearson’s correlation coefficient of 2 h was 0.2384, while the coefficient of 4 h was 0.6281 ( Supporting Information Figure S10c). These results confirm that DNPs colocalized well with early endosomes at ∼2 h and late endosomes/lysosomes at ∼4 h incubation, respectively.

Spatiotemporally controlled intracellular dynamic assembly of DNPs

Two fluorescence resonance energy transfer probes, Cy3-BHQ2-labeled YA and Cy3-BHQ2-labeled YB, were designed to explore the degradation of YA and YB in the endosome/lysosome. CLSM was utilized to observe the changes of fluorescence. When these probes were degraded, the distance between Cy3 and BHQ2 increased; consequently, the intensity of fluorescence signal from Cy3 increased. Compared with the DNP group, the fluorescence intensity of DNP-UV2 group increased and was higher than that in DNP-UV4 group, indicating that the degradation of YA and YB occurred in the stage of endosome/lysosome ( Supporting Information Figure S11). According to the spatiotemporal location of DNPs in living MCF-7 cells, UV irradiation (365 nm, 5 W, 2 min) was implemented at critical time points of 2 and 4 h, respectively. The intracellular assembly of DNPs was observed via CLSM by capturing images at 6 h incubation. The fluorescence signals in DNP-UV2 (UV irradiation at 2 h) and DNP-UV4 (UV irradiation at 4 h) groups were stronger than that of DNP group (cells treated with only DNPs); the red fluorescence signal of DNP-UV4 was the strongest and presented large fluorescent dots as DABs (Figure 3a–d and Supporting Information Figure S12). These phenomena indicated that the assembly from DNPs to bulk DABs could be realized by efficient disassembly and reassembly in late endosomes/lysosomes mediated by UV light. In contrast, when UV light was introduced at the early endosome stage, the size of assemblies formed in late endosomes/lysosomes was smaller, due to the digestion of hydrolases toward disassembled free branches before the reassembly in the late endosomal/lysosomal acidic condition.

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We hypothesized that the intracellular assembly of DNPs would affect autophagy in cells.¹² The assembly of DNPs, formation of DABs, and the changes of autolysosomes were observed by TEM. Autolysosomes were identified as vesicles containing characteristic multilayer structures, that is, multilamellar bodies. The observed nanoparticles and assemblies were determined as DNPs and DABs, respectively, according to the previous literature.^10–12,15 Compared with the PBS group (PBS-treated cells) and the UV group (cells irradiated with UV light without DNPs), the DNP group presented small spheres in autolysosome (Figure 3ei,fi, red arrows, Supporting Information Figure S13); in the DNP-UV2 group, DNPs existed in autolysosome as free DNA fragments and as part of fragments in small assemblies (Figure 3eii,fii, red arrows, Supporting Information Figure S13); in DNP-UV4 group, DNPs assembled into large DABs in autolysosome triggered by H⁺ (Figure 3eiii,fiii, red arrows, Supporting Information Figure S13). In addition to the different assembly states of DNPs, the volume of autolysosomes increased along with the formation of DABs, suggesting that the assembly of DNA nanostructures led to lysosomal swelling.¹¹

Autophagy regulation

Autophagy is a cellular mechanism that removes intracellular redundant objects to maintain normal cell homeostasis.^30,31 We further studied the autophagy of MCF-7 cells regulated by light-mediated intracellular spatiotemporal assembly. The autophagy level was determined by using a Cyto-ID autophagy detection kit, which was specific to phagosomes, autophagosomes, and autolysosomes in living cells.^32,33 PBS, DNP, and UV groups showed weak green fluorescence (GFP) signals, indicating low autophagy levels (Figure 4a). DNP-UV2 (UV irradiation at 2 h) and DNP-UV4 (UV irradiation at 4 h) groups showed obvious fluorescence signals; and the corresponding quantitative analysis showed that the signal intensities of DNP-UV2 and DNP-UV4 groups were 2.2 and 3.0 times of that in the group (Figure 4b), indicating significant enhancement of the DAB-induced autophagic response by the assembly of DNPs. These data show that the controlled formation of intracellular assemblies promotes the level of autophagy and the size of the assemblies affects the level of autophagy.

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To explore the autophagic process during the controlled formation of intracellular assemblies in the DNP-UV4 group, the cells were transfected with Ad-mCherry-GFP-LC3B (adenovirus packaged plasmid).³⁴ The expression of fusion protein mCherry (red fluorescence) and GFP was observed by CLSM to detect the autophagic process. As shown in Figure 4c, before the treatment of DNPs, weak GFP and mCherry signals were observed in the cytoplasm, representing diffused LC3B protein. After treatment with DNPs for 6 h, a large number of yellow puncta (merged by GFP and mCherry fluorescence) appeared in the cells, suggesting the formation of early autophagosomes. Increased numbers of yellow dots were observed in the cells with 8- and 10-h sustained treatment, suggesting that the autophagosomes gradually matured. At the time points of 12 and 24 h, the images show the increase of mCherry puncta and the decrease of GFP signal, reflecting an increased autophagic flux. At the time point of 48 h, the number of both yellow and red puncta were fewer than that at 24 h, indicating that the process of autophagy was weakened.

After the disassembly of DNPs, branched DNA with i-motif terminals were released, which further cross-linked with each other, mediated by H⁺ in lysosomes, leading to the decrease of lysosomal acidity. To assess the lysosomal acidity, a pH-dependent probe, LysoSensor Green, was utilized to stain lysosomes. The fluorescence intensity of the green probe improved as acidity increased. The images showed that the fluorescence intensity of LysoSensor in the DNP-UV2 and DNP-UV4 groups was significantly stronger than that in the PBS group ( Supporting Information Figure S14), indicating that proton-driven assembly significantly decreased lysosomal acidity. As a result, after the fusion of lysosome and autophagosome, the acidity of the formed autolysosome became lower (Figure 4d). The change of pH influenced the activity of acid phosphatase (AP) in autolysosomes, an enzyme that catalyzed the hydrolysis of phosphate esters under an acidic environment. The corresponding enzyme activities in cells at 12 and 24 h were detected by an AP detection kit. The intracellular AP activity showed slight changes in PBS, DNP, and UV groups (Figure 4e and Supporting Information Figure S15). At 12 h, the enzyme activity of DNP, DNP-UV2, and DNP-UV4 groups was 101.0%, 53.4%, and 37.9% of that in PBS group, respectively, and the enzyme activity of the DNP-UV4 group was lower than the DNP-UV2 group (Figure 4e). The enzyme activity of the DNP, DNP-UV2, and DNP-UV4 groups detected at 24 h was 94.2%, 81.8% and 74.8% of that in PBS group, respectively. DNP-UV2 and DNP-UV4 groups were higher than that of 12 h, with no significant difference between the two groups ( Supporting Information Figure S15). We inferred that in the dynamic microenvironment inside cells, the interference in assembly was temporary, and AP activity gradually returned to stabilized states.

Cytoskeleton regulation

Autophagosomes combine with lysosomes to form autolysosomes in cells. The movement of autophagosomes and lysosomes and the occurrence of autophagy depend on actin, and in turn the enhancement of autophagy affects the direction of actin.^33,35–37 As a result, the assembly of DNA nanostructures interfered with the cytoskeleton and further affected the migration of cells (Figure 5a). To evaluate the interference, MCF-7 cells were incubated with DNPs for 24 h after varied treatments, and then the F-actin of cytoskeleton was stained with FITC-labeled phalloidin (green) and observed by CLSM. Long and smooth actin filaments were observed in PBS, UV, and DNP groups, extending throughout the cell bodies (Figure 5b). In contrast, some fragmented F-actin filaments aggregated in MCF-7 cells of the DNP-UV2 group as green fluorescent dots, and more and larger fluorescent dots of fragmented F-actin filaments were observed in DNP-UV4 groups. The direction of F-actin filaments in the cells was further analyzed by using ImageJ software, where the same color represented the same direction in the pattern (Figure 5c). The cell patterns of PBS, UV, and DNP groups showed monotonous color, indicating that the direction of cytoskeleton F-actin was highly consistent. In contrast, DNP-UV2 and DNP-UV4 groups showed multicolor patterns, indicating the reorganization of cytoskeleton F-actin.¹⁵

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The cytoskeleton as a dynamic network of filamentous proteins can support cells and mediate cell movement.^38–40 Thus, cell migration affected by light-mediated assembly of DNPs was investigated next. MCF-7 cells were incubated with DNPs and exposed to UV light at time points of 2 and 4 h, respectively, and cell scratch healing experiments were performed to study cell migration. In the first 24 h, MCF-7 cells in a DNP group showed equal mobility with PBS, UV, DNP-UV2, and DNP-UV4 groups (Figure 5d,e). After 48 h, the difference in scratch healing became significant. At 72 h, the migration speed of MCF-7 cells in the DNP-UV4 group was the slowest, followed by the DNP-UV2 group. At 96 h, the scratch in cells in PBS, UV, and DNP groups closed up. Meanwhile the gap in the DNP-UV2 and DNP-UV4 groups remained, and the scratch in the DNP-UV4 group was the most obvious. To investigate whether the cell migration was caused by the toxicity to cells, cytotoxicity experiments were further performed. To exclude the influence of UV exposure on cell viability, the MTT assay was performed, and the results demonstrated that cell viability was negligibly affected during UV exposure for 2 min ( Supporting Information Figure S16). MCF-7 cells with varied treatments continued to culture for 48, 72, and 96 h, and the cell viability was evaluated by MTT assay. The results showed that the assembly of different concentrations of DNP for varied durations did not affect cell viability in all the groups (Figure 5f and Supporting Information Figure S17), confirming that the assemblies formed in cells led to the influence of cytoskeleton and affected the migration of cells. In addition, the escape kinetics of materials (DNPs or DABs) from cells was studied. TAMRA-labeled DNPs were incubated with MCF-7 cells, and cells were subjected to UV light with varied treatments, continuing to culture for 6, 12, 24, 48, 72, and 96 h, and the escaped materials from cells at these different time points were detected by measuring the fluorescent signals in the supernatant. The relative fluorescence of DNP-UV4 group was lower than that of DNP-UV2 group and DNP group, indicating that the formation of DABs reduced cellular escape of materials ( Supporting Information Figure S18).

Conclusion

In conclusion, we have developed a DNA nanostructure-based system. A logically dual-mode control of spatiotemporally dynamic assembly of DNA nanostructures was realized by both exogenous UV light and endogenous lysosomal acidity, rather than passively driven by the intracellular environment. In living cells, by introducing light at two different time points, the sizes of formed DABs were respectively controlled, which sequentially regulated the autophagy level and affected a series of cell behaviors.

In particular, our reported DNA nanostructures-based assembly system can be expanded to more biological systems through rational molecular design. (1) Due to the programmability of DNA molecules, branched DNA and linker DNA in this system can be designed with more functional sequences such as stimuli-responsive and therapeutics sequences to achieve wider biomedical applications during their spatiotemporal assembly. (2) DNA molecules are quite compatible with many other functional materials, such as proteins and nanoparticles, and thus the assemblies can be endowed with more biological activities, even acting as artificial organelles. (3) By virtue of the library of aptamers, this system can be adapted to a variety of cells by integrating it with varied cell-target aptamers, achieving the exploration of more cell behaviors and biological effects.

Supporting Information

Supporting Information is available and includes materials and partial methods, Table S1, and Figures S1–S18.

Conflict of Interest

There is no conflict of interest to report.

Funding Information

This work was financially supported by the National Natural Science Foundation of China (grant nos. 22225505, 22322407, and 22174097). D.Y. thanks Fudan University Ruiqing Education Funding.