Reagents and materials. All DNAs were synthesized by Sangon Biotech (Shanghai) Co., Ltd, modified oligonucleotides were purified by HPLC, while unmodified oligonucleotides were purified by PAGE. The sequences are listed Table S1. Nucleases and their corresponding buffers were obtained from NEB (Ipswich, MA). Doxorubicin was obtained from Beijing Solarbio Science & Technology Co., Ltd. All chemicals were used as received without additional purification. DNase/RNase-free deionized water (ddH2O) from Tiangen Biotech Co. (Beijing, China) was used in all experiments. Hoechst 33342 was obtained from Solarbio Co., LTD (Beijing, China).
oxDNA simulation. Structural analysis of the different types of the DNA nanotubes were achieved by using oxDNA which is a coarse-grained molecular dynamics (MD) simulation software. We used caDNAno to draw the DNA nanotubes, and export the file formats to “xx.top” and “xx.oxdna” through TacoxDNA. The position and orientation of each base and the overall DNA structure are intuitively presented in a visual image. The image is visualized by the oxDNA generated file in the oxView (https://sulcgroup.github.io/oxdna-viewer/), then we used “RELAX” option to oxDNA interaction, we can modify parameters in the input file, such as the number of steps, temperature and other conditions, and adjust hydrogen bond strength in the file of “oxDNA2_sequence_dependent_parameters” for simulation. The detailed simulation parameters and conditions are noted in Table S2. We used Python files to run the obtained trajectory files and extracted and analyzed the data to obtain the results of RMSF and hydrogen bond occupancy. Then we plotted the data and compared the data of different DNA nanotubes.
Preparation and characterization of DNA nanotubes. All sequences were dissolved and diluted in DNase/RNase-free deionized water. To synthesize tubular DNA, 2.5 μL of 20 μM of DNA sequences (S1-P1, S1-P2, S1-P3, S1-P4, and S1-P5, for sequences see Table S1) were mixed in TM buffer (40mM Tris,1 mM EDTA-Na2, 12.5 mM Mg2+, pH=8). The DNA solutions were heated to 90 °C for 5 min and slowly cooled down to room temperature (1.5h or 18h). The products were characterized by 8% native polyacrylamide gel electrophoresis which was operated at 4 °C for 1 h at a constant voltage of 120 V. The gel was subsequently stained with SYBR Gold dye. Cy5-labelled DNA nanotubes were imaged using TIRF microscope with a TIRF objective (100× magnification, 1.49 NA, Nikon). The lenses, reflection mirrors, and dichroic mirrors were from Semrock (USA). For TIRF illumination, a solid laser of 520 nm was coupled into single-mode fiber cable (Solamere Technologies). Samples containing nanotubes were imaged at 100 nM tile concentration in TM buffer.
Reaction kinetics of the nanotubes to nucleases. 1 μM Cy5 labeled strand (S3-Cy5), 1 μM BHQ3 labeled strand (S4-BHQ3) and 1 μM other strands (S1-P1, S1-P2, and S1-P5) were mixed and annealed in TM buffer to prepare dual-labeled nanotubes. In a typical fluorescence dequenching assay, 250 nM nanotubes, and enzymes were incubated in TM buffer. Once the enzymes were added, fluorescence was recorded immediately in the Cy5 channel (ex: 625 nm, em: 660 nm) of a real-time PCR cycler (Rotor-Gene Q, QIAGEN, Germany) at 37 °C with a time interval of 5 s.
DOX loading and in vitro release. For DOX loading, 500 nM as-prepared nanotubes and 1 mM DOX was mixed with nanotubes and incubated for 24 h at 37 °C and incubated for different time in TM buffer with different pH value. Ultrafiltration (14,000 rpm for 10 min) was applied to remove free DOX. The bound DOX was estimated according to the concentration of free DOX. The drug loading efficiency and loading content were calculated as follow:
Loading efficiency (%) = loaded DOX (mg)/initial DOX (mg) × 100% (1)
Loading content (wt%) = loaded DOX (mg)/nanostructure (mg) × 100% (2)
To obtain DOX release curve, the drug containing nanostructures was prepared in TM buffer at pH 5.6 (endosomal pH) or at pH 7.4 (physiological pH). The solution was shaken at 50 rpm, followed by ultracentrifugation at different time intervals. The absorbance of supernatant at 492 nm was measured on a UV-visible spectrometer (AMR-100, Hangzhou Allsheng Instrument CO., Ltd).
Cell Culture. The HeLa cells, A549 cells and MCF 7 cells were cultured in a RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin under standard conditions (5% CO2, 37 °C). The HEK293T cells were cultured in a DMEM medium with 10% fetal bovine serum and 1% penicillin/streptomycin under standard conditions (5% CO2, 37 °C). The medium was replaced every 24 h, and the cells were digested with trypsin and resuspended in fresh complete medium before plating.
In vitro cellular uptake. The cellular uptake behavior of nanotubes and DOX delivery in cultured cell lines were investigated by using the HILO fluorescence microscopy. According to the cell thickness and the S/N of single-molecule fluorescence, the micrometer-driven optical rail for Z adjustment was adjusted to achieve HILO illumination. The cells were seeded into confocal dishes with a density of 4×105 cells/well and incubated with RPMI 1640 Medium in a humidified atmosphere containing 5% CO2 at 37 °C for 24 h. Then the culture medium was replaced by RPMI 1640 Medium with nanotubes in TM buffer. After 2h of incubation, the mixture in each well was dumped, and washed three times with 1 × PBS. The cell nucleus was stained by Hoechst 33342. The Cy5 fluorescence of nanotubes (ex: 650 nm, em: 670 nm) and DOX fluorescence (ex: 480 nm, em: 550 nm) were recorded by HILO fluorescence microscopy. The Hoechst 33342 (ex: 350 nm, em: 460 nm) was used to stain nucleus.
In vivo tumor therapeutics. BALB/cnude mice were obtained from Servicebio Co., Ltd. (Wuhan, China), and raised in a specific pathogen free animal facility and had access to water and food. The animal experiment protocols were approved by the Animal Ethics Committee of the Beijing University of Chemical Technology. To set up xenograft tumor models, HeLa tumor cells (2 × 106 cells per 100 µL in 1:1 (v/v) PBS and Matrigel, BD bioscience) were inoculated under the armpits of mice until the tumor volume reached 5 mm in diameter. The tumor-bearing nude mice were randomly divided into five groups (n = 5, each group). The mice were then treated three times on 0 day, 3 day, 6 day and 9 day by tail vein injection of PBS, free DOX, control (no uracil)-DOX and S1-P5-DOX (nanotubes equivalent 17.4 mg kg−1 , DOX equivalent 2 mg kg−1). After two-week treatment, the tumor was harvested and analyzed by histological section. The body weight and tumor size (length × width2/2) were measured at 2 days intervals after treatment. After all the experiment were completed, the tumors were collected and weighed. The tumors and major organs of mice were harvested and fixed in 4% paraformaldehyde. After embedded in paraffin, sectioned, and stained with Hematoxylin and Eosin (H&E) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) was performed for histological examination and to assess apoptosis levels in the tumor.