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1 Copyright WILEY VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, Supporting Information for Small, DOI: /smll A Compact DNA Cube with Side Length 10 nm Max B. Scheible, Luvena L. Ong, Johannes B. Woehrstein, Ralf Jungmann, Peng Yin, and Friedrich C. Simmel*
2 Copyright WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, Supporting Information A compact DNA cube with side length 10 nm Max B. Scheible, Luvena L. Ong, Johannes B. Woehrstein, Ralf Jungmann, Peng Yin, and Friedrich C. Simmel* Content S1. cadnano design S2. Why a circular scaffold routing is discarded S3. Magnesium screening S4. Additional TEM images S5. Additional bleaching steps of Cy3 labeled cubes S6. Additional super-resolution images of cube-polymer complexes S7. Sequence list 1
3 S1. cadnano design Cube: X layers are given in dark blue. Y layers are given in the following color code: layer I: red; layer II: cyan; layer III: green; layer IV: yellow. The additional short blue segments on the left and right side support the design-based labeling of Y strands with four thymine nucleotides to prevent base stacking on the blunt ends. 2
4 DNA origami filament monomer (tunnel structure): 3
5 S2. Why a circular scaffold routing of the cube is discarded. A circular scaffold routing is theoretically possible (upper image), but it requires a shift between adjacent layers and therefore distorts the overall cubic structure. Further, the addition of staple strands reveals design-related problems such as too short and unstable domains (red arrows) or blunt ends which lead to undesirable base stacking interactions (green arrows). 4
6 S3. Magnesium screening 5
7 S4. Additional TEM images 6
8 S5. Additional bleaching steps of Cy3 labeled cubes Trace 1 Trace 2 Trace 3 Intensity (a.u.) Time (s) Traces are taken from individual and clearly separated point-spread functions (PSFs) from surface immobilized cube structures. Due to single molecule blinking of the fluorophores many traces rather appear noisy and smeared instead of showing clear bleaching steps. This is why a qualitative display of examples is favored over an incomplete quantitative treatment. 7
9 S6. Additional super-resolution images of cube-filament complexes 120 nm 290 nm 8
10 3 µm Zoom in for details. Single green/red/yellow spots depict fiducial markers for colocalization. 9
11 S7. Gel lane profile Gel electrophoresis of four independent cube folding approaches. The profiles of the gel lanes show two peaks for cube structures (hachured) and unfolded single-stranded DNA, respectively. Yields are determined as ratio of the hachured cube area over the total area. The gel is quantified using ImageJ gel analyzer. The folding yield is determined to approx. (27 ± 2) %. 10
12 S8. Sequence list Cube: Name Sequence Comment SST_4432_X(0,37) GTAAGTTGAAGTAGGAAGCTTTTTCTAGCCATAGCATCGACACTACGACCTGCTTTTCGACAC X Layer 4 SST_4432_X(3,27) GGACTGCATTCTGGACAGTAACTGCATTAACTACGTGCTCCCAACATAAGTGACGTCCTCAGCAG X Layer 4 SST_4432_X(4,16) TTGAAAATTATCTCGATAAGCAGAAGGACCTGTATAACTGGCAAGAGACAAGGCCGCTTCAGAA X Layer 3 SST_4432_X(7,47) AGGATAGCCGGACCGTATTAATGCCGCGCCAACGGTTTCCCGGACCTAGTGTCTATCAAGTCTA X Layer 3 SST_4432_X(8,28) TTCTATGAAACCATTCTCGGGTCGAGCGGGTCACTGTTGTGACCTACGAGAAGCGTATAGATGT X Layer 2 SST_4432_X(11,35) TCCGCGCGAATAGCTCACAGGCGAACTACGTATGAATTGGTTTAAACGCTCCTCGGGAATTAAT X Layer 2 SST_4432_X(12,16) ACGACAGGTGGCAAACCACCTCCGATGTCAGCGCCGCATACCCATTCACTGTGAATTTCCACAC X Layer 1 SST_4432_X(15,47) CGAGGATTCGCAGGTCCATGGGATTCACCAAGCTCGTATACACCCTGATTCTCCATGGCAGCGC X Layer 1 SST_4432_Y(3,12) TTTTAGAATGCAGTCCGTGTCGAAAAGCATAGACACTCG Y Layer IV SST_4432_Y(4,51) TTTTTAGACTTGAGGTCGTATTTT Y Layer IV SST_4432_Y(5,12) TTTTCAGGTCCTTACTGTCCTTTT Y Layer III SST_4432_Y(6,51) TTTTTTGGCGCGGCTTCCTATTTT Y Layer II SST_4432_Y(7,12) TTTTTTCTGAAGCACTTATGTTTT Y Layer I SST_4432_Y(0,51) TTTTCTTCAACTTACCTGCTGAGGACGTCGGCCTTGACGGTCCGGGTTT Y Layer I SST_4432_Y(1,12) TTTTTTGGGAGCACGTAGTTTAGAAAAAGCATTAATTCTCTTGCTAAAC Y Layer II SST_4432_Y(2,51) TTTTGTGTCGATGCTATGGCAATGCAGTTCTGCTTATAGGTCCGGGTCA Y Layer III SST_4432_Y(8,51) TTTTCCGAGAATGCTATCCTTTTT Y Layer I SST_4432_Y(9,12) TTTTGGAGCGTTCAGTTATATTTT Y Layer II SST_4432_Y(10,51) TTTTTTCTCGTAGGAAACCGTTTT Y Layer III SST_4432_Y(11,12) TTTTCTGTGAGCATTTTCAATTTT Y Layer IV SST_4432_Y(4,28) AGATATATTCGCGCGGAACATATGGAGAAGTTTGCCACCTGTCGTTTTT Y Layer IV SST_4432_Y(8,34) CATAGAAATTAATTCACAGGACCTGCGAATCCTCGTTTT Y Layer I SST_4432_Y(9,29) CAACAGTGACCATCCCATGTGAATGGGTATGCGGCTTTT Y Layer II SST_4432_Y(10,34) CAATTCATACGCGGAGGTGTCAGGGTGTATACGAGTTTT Y Layer III SST_4432_Y(12,51) TTTTGCGCTGCCCTATACGCTTTT Y Layer IV SST_4432_Y(13,12) TTTTGCTGACATTAGTTCGCTTTT Y Layer III SST_4432_Y(14,51) TTTTCTTGGTGACGCTCGACTTTT Y Layer II SST_4432_Y(15,12) TTTTGTGTGGAAATTCCCGATTTT Y Layer I Binding domain GTTAGGAGTAGGGTAGTAGG Binding domain for bottom extension Linker strand CCTACTACCCTACTCCTAACTTATACATTGA Linker with 2x P1 including 2x TT spacer TTATACATCTAGTTATACATCTAG P5* 2x P1 extension for cube SST_4432_bt_Y(0,51) TT TTT TCT TCA ACT TAC CTG CTG AGG ACG TCG GCC TTG ACG GTC CGG GTT T 5 -Biotin anchor SST_4432_bt_Y(4,51) TT TTT TTA GAC TTG AGG TCG TAT TTT 5 -Biotin anchor SST_4432_bt_Y(8,51) TT TTT TCC GAG AAT GCT ATC CTT TTT 5 -Biotin anchor SST_4432_bt_Y(12,51) TT TTT TGC GCT GCC CTA TAC GCT TTT 5 -Biotin anchor Imager P1* CTAGATGTAT ATTO 655 Imager P3* GTAATGAAGA Cy3b S2 TAACATTCCTAACTTCTCATA Top extension for selected Y strands S2* TATGAGAAGTTAGGAATGTTA Cy3 DNA origami polymer: The original sequence list including biotin modifications and DNA-PAINT docking sites are published in Ralf Jungmann et al., Multiplexed 3D Cellular Super-Resolution Imaging with DNA-PAINT and Exchange-PAINT. Nature Methods, February 2, 2014, doi: /nmeth
13 Additional sequences: Name Sequence Comment t2_0[199]2[200]_cp5 CCTTACACAGCAAATCGTTTGGGTGGTAAAACTTTCAATGTAT filament2 with t2_2[7]2[8]_cp5 GATTAAATCAGCTCCAATAGGAACGAATTAACCGTCTTCTTTTTTCAATGTAT filament2 poly with TT spacer and t2_3[88]2[72]_cp5 TTCTCCGAGTGGTCAGTTTGGAACAAGAGACATTTCAATGTAT filament2 with t2_18[55]2[40]_cp5 AATCATAATTACAACAAACGCCTAGCCAACGCCACACGACGCTCAATCTTTCAATGTAT filament2 with t2_18[239]2[232]_cp5 AACCAGACCTCTTTAACGCGTCAATCATTAACATTTTACATTTCAATGTAT filament2 with t2_19[168]2[168]_cp5 ACGTTAATTTTAGGAATGTCACTGAGCCAGCGTTTCAATGTAT filament2 with t2_21[136]2[136]_cp5 ACCAGAACGAGTATTAGCAGCGTGCCTGTTCTTTTCAATGTAT filament2 with t2_22[119]2[104]_cp5 TTTTCACGGGCACCAAAGTGGCGAAAATCCTTTTCAATGTAT filament2 with t3_2[103]3[119]_cp5 GTTTGATGAATCGGCAAAATTTGCGTATTGGTTTCAATGTAT filament3 with t3_3[248]3[23]_cp5 TGTTAGTAATAACATCACCCATTTCAATGTAT filament3 poly with TT spacer and t3_5[52]3[55]_cp5 CGCGAACTGATTGGCACAGACAATATATGGAAATTTTCAATGTAT filament3 with t3_6[183]3[183]_cp5 AGTTTTAAGACGATAATCTGGTCACAACCAGCTTTCAATGTAT filament3 with t3_6[247]3[247]_cp5 GCAAATATGCAAAGCGTTTTGTTATAAATTTTTTTCAATGTAT filament3 poly with TT spacer and t3_22[95]3[87]_cp5 AAAAACAGCTTGATACCGATACTTAGCGGGTTGAGTGTTGTTTCAATGTAT filament3 with t3_22[151]3[151]_cp5 ATTGTAAATTGGGCTTCCGTGAGCCTCCTAGCTTTCAATGTAT filament3 with t3_23[216]3[215]_cp5 ATCCCCCTTAAGAGGTCATTTTTGCGGATTACCAGCCGGGTCACTGTTTCAATGTAT filament3 with t2h_2[71]23[71] GAGATAGAGACCTGAAAAATCAAGCTATTTTG filament2helper t2h_2[135]31[135] TCGTTTTCTTTTCACCCCTAAAACAAAGAATAAGCACCATTACAGCGT filament2helper t2h_2[167]5[167] GTGCCGGTGTGGTGCCATCCCACGGCAGCAACCGCAAGAAATGACTTG filament2helper t2h_31[136]19[143] CAGACTGTAGCGCGTTTTCATCGCGATTACCCAAATCAACGTAACATTCAGTGA filament2helper t3h_2[199]23[215] GGCATCAGGGAGGTGTCGAGGCATATAGCGAGAGGCTTATTCATTGA filament3helper t3h_3[24]31[31] GCCATTGCTGGATTATGAACGCGAAGGGCTTAGAACAAAG filament3helper t3h_3[152]20[152] ACCGTCGGCCCCCTAGAACTGCCAGTCA filament3helper t3h_3[184]18[184] TTACGGCTATGCCGGGTACATAACGATTCATCGCCAGCATCCAAGGGT filament3helper t3h_3[216]20[216] TTGAGTAAGAGCGCCCTAAGAGAGGATTATTG filament3helper t3h_5[168]4[184] TAGAACGTCAGCGTGGTGCTGCAGGTCATTGG filament3helper t3h_6[151]22[152] TTTCCCGTTCAACTTTAATCATTTTATGCG filament3helper t3h20[151]21[135] GGAGCTTGCCCTGACGAGAAGGCAGAAAGAAC filament3helper t3h_20[215]21[199] CTGAATATAATGACAGGTAGAAAGCCAAAAG filament3helper t3h_23[184]21[183] AAACCAAAAGTAAGAGCAACACTAAATGCAGA filament3helper 12
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