Supporting Information for. Localized DNA Hybridization Chain Reactions. on DNA Origami

Transcription

1 Supporting Information for Localized DNA Hybridization Chain Reactions on DNA Origami Hieu Bui 1,3*, Shalin Shah 2*, Reem Mokhtar 1, Tianqi Song 1, Sudhanshu Garg 1, John Reif 1,2 1 Department of Computer Science, Duke University 2 Department of Electrical & Computer Engineering, Duke University Durham, NC 27708, USA 3 National Research Council, 500 Fifth Street NW, Keck 576, Washington, DC 2001, USA * These authors contributed equally to the project All correspondence should be addressed to reif@cs.duke.edu 1

2 Detailed design schematic Figure S1: a-c) DNA origami rectangle design. Modified staple strands for tethering DNA hairpins are colored red. Biotin-labeled staple strands are colored green. Modified staple strand for attaching marker sequence is colored blue. Biotin labeled strands are modified so that they extend in the opposite direction of hairpins. Cadnano design file (JSON) attached as supplementary material. Table S1: DNA staples modified to attach biotin labels, hairpins and marker strands. Name Unmodified Staples Modified Staples 5[32]7[31] t5r6f, ACGGCTACAAGTACAACGGAGATTCGCGACCT Biotin-TTTT-ACGGCTACAAGTACAACGGAGATTCGCGACCT 13[96]15[95] t1r14f, AAGAGGAACGAGCTTCAAAGCGAAAGTTTCAT Biotin-TTTT-AAGAGGAACGAGCTTCAAAGCGAAAGTTTCAT 25[192]27[191] t5r26f, CTAAAATAAGTATTAACACCGCCTCGAACTGA Biotin-TTTT-CTAAAATAAGTATTAACACCGCCTCGAACTGA 25[2]27[223] t7r26f, AGATTAGATTTTCCAGCAGAAGATAAAAAATACCGA AGATTAGATTTTCCAGCAGAAGATAAAAAATACCGA-TTTT- AGGTAAGAATGATGTATGTGTGG 10[79]8[80] t3r10e, ACGAACTATTAATCATTGTGAATTTCATCAAG TCTCCTCTCCTCCACTACTCTCAA- ACGAACTATTAATCATTGTGAATTTCATCAAG 9[64]11[63] t3r10f, TTTCAACTACGGAACAACATTATTAACACTAT TTTCAACTACGGAACAACATTATTAACACTAT-TCCTTCTTTCCTTTCCTTCCTTCT 12[79]10[80] t3r12e, ACTGGATATCGTTTACCAGACGACTTAATAAA CACCACCACACCACACCACACCAA-ACTGGATATCGTTTACCAGACGACTTAATAAA 2

3 11[64]13[63] t3r12f, CATAACCCGCGTCCAATACTGCGGTATTATAG CATAACCCGCGTCCAATACTGCGGTATTATAG-TCTCTCCTCTCCTTCTCCTCCTCC 14[79]12[80] t3r14e, TCAGAAGCCTCCAACAGGTCAGGATTTAAATA TCAGAAGCCTCCAACAGGTCAGGATTTAAATA-TTTCTCTCTTCTTCTTCTTTCTTC 13[64]15[63] t3r14f, GAAGCAAAAAAGCGGATTGCATCAATGTTTAG CTTCTCTTTCTTCTTCTTCACTTC-GAAGCAAAAAAGCGGATTGCATCAATGTTTAG Table S2: DNA oligomers for constructing the proposed system. Note: Toehold domains (colored green), stem domains (colored blue), clamp domains (colored red), spacer domains (colored black). For kinetic studies, RC duplex complex is displayed by shaded domain of H6. Name Sequence (5 to 3 ) L Hairpin H1 5 linker GGGTGGGTTGTGATGTATGTAATGTTTTCCTCCTACTCACATACATCACAAC 3 52 Hairpin H2 5 ACTCACATACATCAATCGTCTTTCTTTTGTTGTGATGTATGTGAGTAGGAGG 3 linker 52 Hairpin H3 5 linker GAAAGACGATTGATGTATGTGAGTTTTTTCTTCCTTTAACATACATCAATCG 3 52 Hairpin H4 5 TTTAACATACATCACCACTTTCCTTTTTCGATTGATGTATGTTAAAGGAAGA 3 linker 52 Hairpin H5 5 linker AGGAAAGTGGTGATGTATGTTAAATTTTCTTCTCCTCCACATACATCACCAC 3 52 Hairpin H6 5 -CTCCACATACATCATATTCCCTCATTCAAGCGTGGTGATGTATGTGGAGGAGAAG 3 linker 55 RC FQ 5 TTGAATGAGGGAATATGATGTATGTGG 3 /Quencher/ 27 RC TET /Fluorophore/ 5 CCACATACATCATATTCCCT 3 20 RC Signal 5 CCACATACATCATATTCCCTCATTCAA 3 27 Initiator I1 5 CATTACATACATCACAACCCACCC 3 Anchor A1 5 TTGAGAGTAGTGGAGGAGAGGAGA H1 Anchor A2 H2 AGAAGGAAGGAAAGGAAAGAAGGA 3 Anchor A3 5 TTGGTGTGGTGTGGTGTGGTGGTG H3 Anchor A4 H4 GGAGGAGGAGAAGGAGAGGAGAGA 3 Anchor A5 5 GAAGTGAAGAAGAAGAAAGAGAAG H5 3

4 Anchor A6 H6 GAAGAAAGAAGAAGAAGAGAGAAA 3 Staple L1 5 TCTCCTCTCCTCCACTACTCTCAA-ACGAACTATTAATCATTGTGAATTTCATCAAG 3 56 Staple L2 5 TTTCAACTACGGAACAACATTATTAACACTAT-TCCTTCTTTCCTTTCCTTCCTTCT 3 56 Staple L3 5 CACCACCACACCACACCACACCAA-ACTGGATATCGTTTACCAGACGACTTAATAAA 3 56 Staple L4 5 CATAACCCGCGTCCAATACTGCGGTATTATAG-TCTCTCCTCTCCTTCTCCTCCTCC 3 56 Staple L5 5 CTTCTCTTTCTTCTTCTTCACTTC-GAAGCAAAAAAGCGGATTGCATCAATGTTTAG 3 56 Staple L6 5 TCAGAAGCCTCCAACAGGTCAGGATTTAAATA-TTTCTCTCTTCTTCTTCTTTCTTC 3 56 I1-TAM 5 CATTACATACATCACAACCCACCC 3 / TAM/ O6-FAM /FAM/ 5 TTGAATGAGGGAATATGATGTATGTGG 3 27 M-ATTO 5 CCACACATACATCATTCTTACCTA 3 /ATTO647/ Figure S2: AFM characterization of DNA origami rectangle labeled with DNA hairpins at thermal equilibrium. Six metastable DNA hairpins self-assembled on DNA origami rectangle (a), after adding the initiator (b), and after adding the biotin-labeled output to detect the reaction completion (c). 4

5 Figure S3: Control AFM characterization of DNA origami rectangle labeled with DNA hairpins at thermal equilibrium. Six metastable DNA hairpins self-assembled on DNA origami rectangle (a), after adding the biotin-labeled output without the initiator (b), and after adding the biotin-labeled output with the initiator(c). Note: N is the total number of all origami structures, d is the total number of origami structures with defects or multimers, 1 is the total number of target structures (origami with hairpins), and b is the total number of target structures with biotin-streptavidin. 5

6 Figure S4: Effect of using fluorescence microscope in TIRF mode. An incident laser beam travels from denser medium (immersion oil) to rarer medium (imaging buffer) and, therefore, undergoes total internal reflection if the incident laser beam is bent higher than the critical angle. This generates exponentially decaying evanescent wave-front at the surface. This effect is shown using the intensity of solution containing red spots. 6

7 Figure S5: Quenching of ATTO 647N labeled marker strand. Blue curve indicates fluorescence of ssdna labeled with ATTO 647N while the orange curve indicates increased fluorescence in presence of complementary DNA strand due to increased mean distance between dye and guanine bases. This leads to a substantial decrease in the amount of PET quenching due to electron donors such as guanine. 7

8 Figure S6: The effect of surface passivation by adding blocking agents BSA and Tween 20. a) On the top is labeled free DNA in 1x PBS. The figure on the bottom shows significant improvement in surface passivation after using BSA and Tween 20. Each data point was collected using 6 different ROI. Scale bars are 5 µm. 8

9 Figure S7: Time series showing reaction growth for DNA origami with hairpins incubated with 10x initiator for 10, 60 and 120 minutes. After incubation, excess labeled strands were removed using centrifugal filtration to minimize background noise and nonspecific binding. For each data point in the plot, N indicates the detected marker spots considered for quantitative analysis. 9

10 Figure S8: Control experiment for TIRF demonstrating a key difference between reaction with and without initiator observed after 200 minutes. For each data point in the plot, N indicates the detected marker spots considered for quantitative analysis. 10

11 Figure S9: Simulating growth curve for localized hybridization chain reaction for 800 minutes at 0x, 2x and 10x initiator concentrations. 11

12 Figure S10: Effect of localized cascade DNA hybridization chain reaction rate as a function of initiator concentration. At the end of each experiment, an addition of 10x initiator I6 strand (with domains S6 C6 R C5) was added to directly bind to the last hairpin H6. 12

13 Visual DSD code (* 6-hairpin system ran for 800 minutes *) directive sample directive simulation deterministic directive polymers directive plot reporter() (* DNA origami concentration is 5 nm *) directive localconcentrations [(a, 1); (b, 1); (c, 1); (d, 1); (e, 1)] (*Adopting rates from Zhang et. al. (2009) and Qian et. al. (2011) *) directive leak 1.0E-8 directive tau 1.0 (* Added N * x amout of intiator at 500 s *) directive sweep input = {conc = [0.0, 2.0, 10.0]} directive event initiator() 500 (* 1x = 5 nm *) def N = 5.0 def initiator() = <C0^ R C1^ S1^> def RC() = <L^*>[S7^* C7^* R* C6^*] def reporter() = <C6^ R C7^ S7^> def origami() = [[ (*hairpin 1*) <tether(a) T1* S1^*>[C1^* R*]{C2^ S2^ L^ C0^*> (*hairpin 2*) 13

14 {tether(a, b) T2* S2^*}[C2^ R]{C1^* L^ S3^ C3^> (*hairpin 3*) <tether(b, c) T3* S3^*>[C3^* R*]{C4^ S4^ L^ C2^*> (*hairpin 4*) {tether(c, d) T4* S4^*}[C4^ R]{C3^* L^ S5^ C5^> (*hairpin 5*) <tether(d, e) T5* S5^*>[C5^* R*]{C6^ S6^ L^ C4^*> (*hairpin 6*) {tether(e) T6* S6^*}[C6^ R]{C5^* L^ S7^ C7^> ]] ( 0 * initiator() N * origami() (* 6.5 nm reporter *) 1.3 * N * RC() ) 14