Programming cell adhesion for on-chip sequential Boolean logic

Transcription

1 Supporting Information Programming cell adhesion for on-chip sequential Boolean logic functions Xiangmeng Qu,, Shaopeng Wang,, Zhilei Ge,, Jianbang Wang, Guangbao Yao, Jiang Li, Xiaolei Zuo, Jiye Shi, Shiping Song, Lihua Wang, Li Li,, Hao Pei*, and Chunhai Fan,*, 1 Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai, , P. R. China 2 Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai , P. R. China 3 Kellogg College, University of Oxford, Oxford, OX2 6PN, UK S1

2 Experimental Procedures Materials and methods DNA oligonucleotides were purchased from Takara (purified by HPLC). SMCC and (3-Aminopropyl)-triethoxysilane (APTES) were purchased from Sigma. Cyclic peptide RGDfK-NH 2 was purchased from Peptides International, Inc. Biotin-PEG-SVA (MW 5,000) and mpeg- SVA (MW 5,000) were purchased from Laysan Bio, Inc. PBS was prepared from NaCl 137 mm,kcl 2.7 mm,na 2 HPO 4 10 mm,kh 2 PO 4 2 mm (ph=7.4). All solutions were prepared with deionized water. Hela cells were cultured in MEM medium supplemented with 10% fetal bovine serum, penicillin/streptomycin (100 units/ml) and L-glutamine (2 mm) at 37 O C in humidified environment containing 5% CO 2. Preparation of pegylated coverslip Glass coverslip was modified with PEG as previously described with a little modification. Briefly, glass coverslip was first cleaned with piranha solution (5% NH 3 H 2 O and 14% H 2 O 2 ), and followed by aminosilanization with 5% APTES. Then N-hydroxysuccinimide (NHS) ester modified PEG (a mixture contain 95% mpeg-sva and 5% biotin-peg-sva) was coupled with amines on the coverslip in 10 mm NaHCO 3. After extensive washing with PBS, 200 µg/ml streptavidin was incubated on biotin coated coverslip for 30 min at room temperature. S2

3 RGD-DNA assemble on coverslip RGDfK-NH2 and thiol modified DNA (release-rgd)were coupled through SMCC (a hetero-bifunctional crosslinker). After hybridization with 5' biotin labeled complementary DNA strands (OR-biotin, AND-biotin, XOR-biotin) in PBS buffer at 37 O C 30 min, 3 µl 1 µm solution was added on streptavidin modified coverslip for assembly. Cell experiment For cell adhesion, coverslips were placed into a 24 well plate, and cells per well were seeded. After an hour, cell culture medium was changed to remove non-adhesive cells. Cells were allowed to spread for another hour, and images were captured by microscope. Different DNA trigger strands were then added at a concentration of 1 µm. After 20 min, cells were gently washed and bright-field and fluorescence images were recorded. For FACS analysis of released cell number, after the addition of corresponding DNA strands for 20 min, the suspension was collected together with the washing buffer after washing the coverslip twice. Cells were then collected by centrifugation and resuspended into 100 µl medium for FACS analysis. Ca 2+ imaging Cells were loaded with 5 µm Fluo-4 AM in the dark at 37 o C in PBS. After incubation for 0.5 h, cells were washed three times with PBS. Before recording, cells were kept S3

4 in the dark for 20 min to allow de-esterification of the dye. Ca 2+ imaging was conducted at 37 o C on an inverted fluorescence microscope (Nikon Eclipse Ti), and the fluorescence signals were quantitatively analyzed with the MaxIm DL software. Table S1. DNA sequences used in this paper are as following. R-RGD OR-biotin SH-TTTTTTTTTTTTTTTGGGAGTATTGCGGAGGAAGGTATATC TCTC Biotin-TTTTTTTTTTTTTTTGTCCTCCAGAGAGATATACCTTCCT CCGCAATACTCCCCAAAGTTG OR- IN1 OR- IN2 AND-biotin Cy3-GGGAGTATTGCGGAGGAAGGTATATCTCTCTGGAGGAC FITC-CAACTTTAGGGAGTATTGCGGAGGAAGGTATATCTCTC Biotin-TTTTTTTTTTTTTTTGTCCTCCAGAGAGATATACCTTCGT GTCTCCGCAATACTCCCCAAAGTTG AND-IN1 AND- IN2 XOR-biotin XOR- IN1 XOR- IN2 AND-OR-1 AND-OR-IN1 Cy3-ACGAAGGTATATCTCTCTGGAGGAC FITC-CAACTTTAGGGAGTATTGCGGAGAC Biotin-TTTTTTTTTTTTTTTCCTTCCTCCGCAATACTCCCGTCCTC CA Cy3-GAGATATACCTTCCTCCGCAATACTCCCGTCCTCCA FITC-TGGAGGACGGGAGTATTGCGGAGGAAGGTATATCTC ATCGATCG CATGAAGGCTCGACTCGCCG CGGCGAGTCGAGCCTTCATGCGATCGAT AND-OR-2 AND-OR-IN2 CCTTCCTCCGCAATACTCCCCGGCGAGTCGAGCCTTCATG CGACTCGCCGGGGAGTATTGCGGAGGAAGG S4

5 Figure S1. Induction of a transient Ca 2+ concentration increase by RGD-coated coverslip surfaces binding to HeLa cells. a) Fluorescence images of Hela-cells loaded with the Ca 2+ indicator Fluo-4 AM, with white circle designating the single cell that was monitored. b) Single-cell Ca 2+ response at indicated times after application of the RGD-coated coverslip surfaces. A key for the heatmap is on the bottom. c) Time course of Fluo-4 fluorescence changes revealing a biphasic, transient Ca 2+ concentration increase in a single cell. F 0 is referred to the fluorescence at initial state, representing the basal Ca 2+ concentration. S5

6 Figure S2. The kinetics of on-chip cell release was drastically accelerated as the toehold length was varied from 4-base to 6-base. Figure S3. An AND operation via toehold-mediated SDR for programmably regulating on-chip cell adhesion and detachment. Specifically, an AND logic gate uses strand a b (IN1) and strand c d (IN2) as inputs. Upon the addition of 1 µm Cy3-labeled IN1 (input=1/0) into the cell culture medium for 20 min followed by the intensive washing with PBS, the IN1 hybridized to the toehold on the 3 -end of the base strand and partially displaced only one duplex region (b ) via a three-way branch migration mechanism, leaving the RGD-coupled ssdna anchored to the base strand. Likewise, the addition of 1 µm FAM-labeled IN2 (input=0/1) bound to the d toehold on the 5 -end of the base strand and proceeded to invade backwards, as a result the RGD-coupled ssdna remained wired to the base strand. In these two operations (input=1/0 and 0/1), the cells remained adherent to coverslip surfaces with only few released into the solution (output=0), similar to the result in which neither input was applied (input=0/0). However, when both IN1 and IN2 were added to the system (input=1/1), the cooperative hybridization of two partially complementary strands (IN1 and IN2) with the base strand displaces the RGD-coupled ssdna by three-way branch migration and triggered the cell detachment (output=1). S6

7 Figure S4. The overall AND operation could be verified using polyacrylamide gel electrophoresis (PAGE) image. Lane 1: 20bp marker; Lane 2: C strand; Lane 3: D strand; Lane 4: input strand a b (IN1); Lane 5: input strand c d (IN2); Lane 6: double strands (DS) formed by R-RGD (b c ) and AND-biotin (abcd); Lane 7: DS and AND-IN1 mixture with equal molar ratio for 2 hours; Lane 8: DS and AND AND-IN1 mixture with equal molar ratio for 2 hours; Lane 9: mixture of DS, AND-IN1 and AND-IN2 with equal molar ratio for 2 hours. Figure S5. An OR operation via toehold-mediated SDR for programmably regulating on-chip cell adhesion and detachment. Specifically, an OR logic gate uses strand a b c (IN1) and strand b c d (IN2) as inputs. The addition of IN1 (or IN2) binds to base strand via invading a toehold on the 3 -end (or the d toehold on the 5 -end) and displaces the RGD-coupled ssdna by branch migration, thus triggering the cell detachment from coverslip surfaces (input=1/0 or 0/1; output=1). When both inputs were present (input=1/1), the output of our DNA-based OR gate was similar to a single equivalent even in the presence of excess inputs (output=1). S7

8 Figure S6. The overall OR operation could be verified using PAGE image. Lane 1: 20bp marker; Lane 2: R-RGD (b c ) strand; Lane 3: OR-biotin (abcd) strand; Lane 4: input strand OR-IN1; Lane 5: input strand OR-IN2; Lane 6: double strands (DS) formed by R-RGD (b c ) and OR-biotin (abcd); Lane 7: DS and OR-IN1 mixture with equal molar ratio for 2 hours; Lane 8: DS and OR-IN2 mixture with equal molar ratio for 2 hours; Lane 9: mixture of DS, OR-IN1 and OR-IN2 with equal molar ratio for 2 hours. Figure S7. An XOR operation via toehold-mediated SDR for programmably regulating on-chip cell adhesion and detachment. Specifically, an XOR logic gate uses strand a b c (IN1) and strand abc (IN2) as inputs. Here, the length of the base strand (strand ab) is shorter than that was used in AND and OR gate (strand abcd). The addition of IN1 bound to base strand via invading the a toehold on the 3 -end and displaced the RGD-coupled ssdna by branch migration, thus inducing the cell detachment from coverslip surfaces (input=1/0, output=1). Whereas, the addition of IN2 bound to the RGD-coupled ssdna via invading the c toehold on the 3 -end and then dissociated the RGD-coupled ssdna from coverslip surfaces, resulting in the cell detachment (input=0/1, output=1). S8

9 Figure S8. The overall XOR operation could be verified using PAGE image. Lane 1: 20bp marker; Lane 2: R-RGD (b c ) strand; Lane 3: XOR-biotin strand; Lane 4: input strand XOR-IN1; Lane 5: input strand XOR-IN2; Lane 6: double strands (DS) formed by R-RGD (b c ) and XOR-biotin (ab); Lane 7: DS and XOR-IN1 mixture with equal molar ratio for 2 hours; Lane 8: DS and XOR-IN2 mixture with equal molar ratio for 2 hours; Lane 9: mixture of DS, XOR-IN1 and XOR-IN2 with equal molar ratio for 2 hours; Lane 10: 20bp marker. Figure S9. An AND-OR operation via toehold-mediated SDR for programmably regulating on-chip cell adhesion and detachment. A two-layer logic circuit with the first layer composed of an AND logic gate using strand 1 2 (IN1) and strand 23 (IN2) as inputs, and the second layer composed of an OR logic gate using the output from the first layer and strand b c (IN3) as inputs. The addition of single-stranded inputs (IN1: strand 1 2, IN2: strand 23) to a solution containing the AND gate initiates a computation. The upstream AND gate was designed such that the addition of IN1 displaces only one duplex region (2), leaving the output strand a b sequestering; whereas the addition of IN2 could not invade the toehold region of strand 23 because strand 12 occupies the toehold and prevents input B from displacing the output strand a b. S9

10 Figure S10. The overall AND-OR operation could be verified using PAGE image. Lane 1: 20bp marker; Lane 2: R-RGD strand; Lane 3: OR-IN1 strand; Lane 4:OR-IN2 strand; Lane 5:double strands formed by AND-OR-1, AND-OR-2 and OR-IN1 (E); Lane 6: E mixed with equal molar ratio of AND-OR-1 for 2 hours; Lane 7: E mixed with equal molar ratio of AND-OR-IN1 and AND-OR-IN2 for 2 hours; Lane 8: double strands formed by R-RGD and OR-biotin (DS); Lane 9: mixture of DS and OR-IN2 with equal molar ratio for 2 hours; Lane 10: mixture of DS and E with equal molar ratio for 2 hours; Lane 11: mixture of DS and E+ AND-OR-IN1 with equal molar ratio for 2 hours; Lane 12: mixture of DS and E+ AND-OR-IN1 + AND-OR-IN2 with equal molar ratio for 2 hours; Lane 13: mixture of DS and E+ AND-OR-IN1 + AND-OR-IN2+ OR-IN2 with equal molar ratio for 2 hours. S10