Spatiotemporal patterning of photoresponsive DNA-based hydrogels to tune local cell responses.

Materials Ultrapure water with 18.2 mΩ•cm was used in all experiments (Heal Force water purification system). All chemical reagents were analytical grade and used without further purification. All DNA oligonucleotides used were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China) and Hippo Biotechnology Co., Ltd. (Beijing, China) and purified by HPLC. The DNA sequences are shown in Supplementary Table 1 -Supplementary Table 4 . HeLa cells used in all experiments were obtained from ATCC (ATCC Number: CCL-2). 3-(Trimethoxysilyl) propyl methacrylate was purchased from Sigma-Aldrich Co., Ltd. Acrylamide, ammonium persulfate (APS) and TEMED were purchased from Bio-Rad Co., Ltd. Calcein-AM and propidium iodide (PI) were obtained from Yeasen Biotech Co., Ltd. (Shanghai, China). The buffer used in all experiments was TE buffer (20 mM Tris-HCl, 150 mM NaCl, and 5 mM MgCl 2 ; pH 7.4).

Hydrogels chamber preparation

The chamber is composed of two glass slides. A spacer (polyimide tape) was mounted on a fluorine resin coated glass slide and the respective pre-gel mixture was placed within the spacer domain and covered with a counter silanized glass slide. The top silanized glass slide served as a substrate onto which the hydrogel samples adhered after polymerization. The bottom fluorine resin coated glass slide prevented the DNA gel from sticking to the glass surface and allowed for minimal edge roughness. For the preparation of fluorine resin coated glass slide, the glass slide was treated with O 2 plasma for 1 min to make the glass surface hydrophilic. The treated glass slide was immersed in a solution containing fluorine resin solution (Item NO: SUREC 2101 S, donated by AGC Chemicals) at room temperature, followed by lifting at a rate of 16 mm/s. Next, the glass slide was dried at room temperature and baked at 120 °C for 30 min. For the preparation of silanized glass slide, the glass slide was sonicated in 10% (w/w) NaOH solution for 30 min, rinsed with MilliQ water, and dried under N 2 gas. The cleaned glass slide was, then, incubated in 4% (v/v) 3-(Trimethoxysilyl) propyl methacrylate isopropanol solution with 0.2% (v/v) acetic acid for 2 h. The resulting modified glass slide was rinsed with absolute isopropanol, and dried with N 2 gas.

Preparation of pre-gel solution

The gel was prepared by free radical copolymerization of acrylamide along with a DNA duplex as the crosslinker. To enable their copolymerization with acrylamide, all DNA strands were modified at the 5’-end with an acrydite unit. The pre-gel solution was prepared by mixing 40% (w/w) acrylamide, initiator (10 mg APS was dissolved in 95 µL TAE buffer with 5 µL TEMED), crosslinker DNA, photocleavable single-stranded DNA and photocleavable hairpin DNA. The final concentrations of all pre-gel components are as follows: 1 mM crosslinker DNA (1 or 4), 10% (w/w) acrylamide, 200 µM photocleavable single-stranded DNA (2) or 200 µM photocleavable hairpin DNA (3) and 0.11 M APS. Formation of photoresponsive DNA-based hydrogels Gels were formed by adding pre-gel solution into the hydrogel chamber for free radical polymerization. Thus, DNA hydrogels with controllable thickness were prepared on the silanized glass slide surface. After gel formation, the gel-coated glass slide was transferred into the TE buffer and incubated in TE buffer overnight to remove the fluorine resin coated glass. In this way, the hydrogels with desirable thickness were fabricated on the 3-(Trimethoxysilyl) propyl methacrylate silanized glass surface. Photopatterning process Photomask based patterning was performed through programmable UV irradiation using designed photomask with desired patterns. The hydrogel film was irradiated by UV light (365 nm, 15 mW/cm 2 ) for 5 min through photomask. All the two-photon lithography procedures were performed by using LSM 880 confocal microscope (Carl Zeiss) equipped with a FemtoSecond Laser (Coherent Inc.). A 40 × objective and 740 nm FemtoSecond Laser with 8% power were used in whole lithography process. After irradiation, the staining solution, 100 µM (2’) or (3’), was added to develop the patterns. The patterned hydrogels were observed using confocal microscope (Carl Zeiss, LSM 880).

Scanning electron microscope imaging

SEM images were taken with Extra High-Resolution Scanning Electron Microscope (Hitachi SU8010) at 10 kV. The patterned hydrogels on glass surface was frozen by immersing it in liquid nitrogen. The frozen sample was dried by sublimation of the formed ice under high vacuum, and further metal-coated with Au. Site-specific swelling of the patterned hydrogels For the hydrogel pattern modulation via site-specific DNA HCR-directed programmable expansion, polyacrylamide/DNA hydrogel was prepared by using pre-gel solution containing 500 μΜ strand (5), 500 μΜ strand (5’), 200 µM strand (2), 200 µM strand (3) and 10% (w/w) acrylamide. After gel formation, the hydrogel was photopatterned and imaged using confocal microscope. Subsequently, 100 μM H1 and H2 mixture was added to the patterned hydrogel and incubated overnight to perform DNA HCR-directed programmable expansion process and the swollen hydrogel was imaged using confocal microscope under 25 °C.

Cell culture on MUC-1 aptamer patterned hydrogel surface

For the cell culture experiment, MUC-1 aptamer patterned hydrogels was prepared according to the following procedure. The hydrogel was prepared using pre-gel solution containing 1 mM strand ( 4 ), 200 µM strand ( 3 ) and 20% (w/w) acrylamide. After gel formation, the hydrogel was photopatterned and modified with the MUC-1 aptamer tailed strand ( 3” ). Subsequently, about 1×10 7 HeLa cells (obtained from ATCC) in 1 mL cell culture medium were seeded on the patterned surface. It should be noted that in contrast to the different patterned hydrogels in the study 10% w/w acrylamide hydrogel, we observed that this composition of the hydrogel is unstable in the presence of the cell-medium growth. Accordingly, for the cell-patterned hydrogels we applied a 20% w/w acrylamide hydrogel. The cells were further cultured in 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 0.5 mg mL −1 penicillin-streptomycin at 37 °C, using a humidified 5% CO 2 incubator. The cultured HeLa cells were stained with 4 μM calcein-AM and 4 μM propidium iodide (PI) for 30 min to verify the viability of the cells. Cultured cells were observed at different time intervals using confocal microscope.

Cell culture on MUC-1 aptamer patterned orthogonal ellipsoid structures

The hydrogel film was prepared by using pre-gel solution containing 500 μΜ strand ( 5 ), 500 μΜ strand ( 5’ ), 200 µM strand ( 3 ) and 20% (w/w) acrylamide. After gel formation, the hydrogel film was photopatterned using MUC-1 aptamer tailed strand ( 3” ) to obtain the circular patterned domains. The patterned hydrogel film was seeded with the HeLa cells, and allowed to cell proliferate while subjecting the continuous hydrogel to the HCR process with the addition of H1 and H2 into the cell culture medium for a time interval of ten hours. Cultured cells were imaged at different time intervals using confocal microscope.

Rheological measurements

Rheological properties were determined by a DHR-2 rheometer (TA Instruments) equipped with a 20 mm parallel plate geometry with a gap size of 0.2 mm. The experimental temperature was fixed at 37 °C by a cyclic water bath. The time sweep measurements were performed at a 1% strain with a fixed frequency of 1 Hz.

Statistics and reproducibility

It should be noted that for the photopatterned images shown for the different systems, no noticeable difference between N = 3–4 experiments could be identified. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Materials Ultrapure water with 18.2 mΩ•cm was used in all experiments (Heal Force water purification system). All chemical reagents were analytical grade and used without further purification. All DNA oligonucleotides used were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China) and Hippo Biotechnology Co., Ltd. (Beijing, China) and purified by HPLC. The DNA sequences are shown in Supplementary Table 1 -Supplementary Table 4 . HeLa cells used in all experiments were obtained from ATCC (ATCC Number: CCL-2). 3-(Trimethoxysilyl) propyl methacrylate was purchased from Sigma-Aldrich Co., Ltd. Acrylamide, ammonium persulfate (APS) and TEMED were purchased from Bio-Rad Co., Ltd. Calcein-AM and propidium iodide (PI) were obtained from Yeasen Biotech Co., Ltd. (Shanghai, China). The buffer used in all experiments was TE buffer (20 mM Tris-HCl, 150 mM NaCl, and 5 mM MgCl 2 ; pH 7.4).

Supplementary information Supplementary Information Peer Review File Description of Additional Supplementary Files Supplementary Movie 1 Supplementary Movie 2 Supplementary Movie 3 Reporting Summary