Supporting Information DNA Origami Nanoantennas with over ...
Supporting Information
DNA Origami Nanoantennas with over 5000fold Fluorescence Enhancement and Single-Molecule Detection at 25 µM Anastasiya Puchkova, Carolin Vietz, Bettina Wünsch, Enrico Pibiri, Maria Sanz Paz, Guillermo P. Acuna* and Philip Tinnefeld Institute for Physical & Theoretical Chemistry, and Braunschweig Integrated Centre of Systems Biology (BRICS), and Laboratory for Emerging Nanometrology (LENA), Braunschweig University of Technology, 38106 Braunschweig, Germany. *Correspondence to: [email protected]
Sample preparation of DNA origami pillar DNA origami pillar was designed using the CaDNAno software (http://cadnano.org/) (1). For the folding of this structure 10 nM of 8064 nt long single stranded DNA scaffold (M13mp18) and 100 nM of each staple strand (see table S1, purchased from Eurofins Genomics) were mixed in a TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0) containing 14 mM MgCl2. The folding mixture was then annealed following the program presented in table S2. After folding the sample was purified from excess staple strands by filtering in Amicon columns (Amicon Ultra – 0.5 ml, Ultracel®- 100K Membrane, Millipore) with TAE buffer (40 mM Tris, 2 mM EDTA) containing 12 mM MgCl2. The sample was centrifuged 3 times at 16 krcf speed for 5 min at 20°C with buffer replacement after each step. To remove purified origami structures the filter column was flipped into a new tube and centrifuged for 3 min at 1 krcf speed.
S1
Functionalization of gold nanoparticles Gold nanoparticles of 100 nm diameter (BBI solutions) were functionalized with 20T DNAoligonucleotides containing a thiol modification on the 3’ end (Ella Biotech GmbH) following the procedure adapted from Mirkin (2) with minor changes. The nanoparticle oligonucleotide mixture was stirred at 40 °C during the salting procedure. By stepwise adding 1xPBS buffer containing 3.3 M NaCl a final concentration of 750 mM NaCl was reached. For purification from free oligonucleotides the nanoparticle solution was spinned down, the supernatant was pipetted off and the nanoparticle pellet was diluted in 1xPBS containing 10 mM NaCl, 2.11 mM P8709, 2.89 mM P8584, 0.01 % Tween20 and 1 mM EDTA-S. This purification step was repeated 7 times.
Figure S1. “Immobilized reference” refers to the quenching of the fluorophore bound to the DNA origami and immobilized on the surface of a coverslip. Therefore the fluorescence intensity of single ATTO647N was determined at different concentrations of NiCl2 as indicated and referred to the reference in the absence of quencher.
S2
Figure S2. Fluorescence quenching of ATTO647N by NiCl2 on the ensemble level measured in a spectrometer. ATTO647N 3’-labelled oligonucleotides with the following sequence were used: GCG CAT CAT CTG CCA GTT TTC ACC AGG CGT GAG CAA GTC TTG
S3
Figure S3. Photon count rate histogram for the dimer nanoantennas with an ATTO655 fluorophore (red) and an ATTO647N with a NiCl2 concentration of 0.5 mM (black). The enhancement is calculated based on the comparison with a reference that consist of an immobilized DNA origami pillar with the same fluorophore and quencher conditions in the absence of any plasmonic interaction.
S4
Table S1. List of unmodified and modified staples from the 5’ to the 3’ end for the DNA origami pillar. Sequence (5’ to 3’)
Length
Modification
TTAGTTTGAGTGCCCGAGAAATAAAGAAATTGCGTAGAGATA
42
TATGACTTTATACATTTTTTTTTAATGGAAACAGTACACCGT
42
AATAAAACGAACTATGACCCCACCAAGC
28
CTCATCGGGATTGAGTGAGCGAGTAACAACCCGTC
35
CCCAGCTACAATGACAGCATTTGAGGCAAGTTGAGAAATGAA
42
TCATACATTTAATACCGATAGCCCTAAAACATCGAACGTAAC
42
ACGCGGTCCGTTTTTGGGTAAGTGA
25
CTTACGGAACAGTCAGGACGTTGGGAAGAAA
31
GGAACCATACAGGCAAGGCAAATCAAAAAGACGTAGTAGCAT
42
CTAAATCGGTCAGAATTAGCAAAATTAAGCAATAAAATAATA
42
TTTAGCGATACCAACGCGTTA
21
AAGAAAGCTTGATACCGCCACGCATACAGACCAGGCGCTGAC
42
CCCCGCTAGGGCAACAGCTGGCGAAAGGGGGATGTGCTTATT
42
GCCCGAGTACGAGCCGGAAGC
21
CATTTCGCAAATGTCATCTGCGAACGAGAGATTCACAATGCC
42
TATTTAAATTGCAGGAAGATTG
22
AAAGATTACAGAACGGGAGAAGGAAACGTCACCAATGAAACCA
43
ACGTAAGAATTCGTTCTTAGAAGAACTCAAACTATCGGATAA
42
GCGAATCAGTGAGGCCACCGAGTAGTAGCAACTGAGAGTTGA
42
TCGTGCCGGAGTCAATAGTGAATTTGCAGAT
31
TAGCCTCAGAGCATACCCTGT
21
AGCAACAAAGTCAGAAATAATATCCAATAATCGGCTCAGGGA
42
AGGCTTGCGAGACTCCTCAAGAGAAAAGTATTCGGAAC
38
ACCTGACGGGGAAAGCCGGCGAACCAAGTGTCTGCGCGTTGC
42
CCGTAATCAGTAGCGACAGAATCTAATTATTCATTAAAAAGG
42
GAACTGGCTCATTACAACTTTAATCATTCTTGAGATTACTTA
42
GAGTTAAAAGGGTAATTGAGCGCTAATATCAGAGGAACTGAACACC
46
AGTTTCCAACATTATTACATTATAC
25
ACGCGAGAGAAGGCCATGTAATTTAGGCCAGGCTTAATTGAGAATCGC
48
ACTAATGCCACTACGAATAAA
21
CTGTATGGGATTACCGTTAGTATCA
25
TAGCCAGCTTTCATCCAAAAATAAACGT
28
CCTCGTTTACCAGAAACCAAA
21
TTAGCCCTGACGAGAAACACCAGAAATTGGGGTGAATTATTTTAA
45
TGAGTAAAGGATAAGTTTAGCTATATCATAGACCATTAGATA
42
ATTTCCTGATTATCAGATGATGGCTTTAAAAAGACGCTAAAA
42
CCAGCCTCCGATCCTCATGCCGGA
24
ATGAAGGGTAAAGTTCACGGTGCGGCCATGCCGGTCGCCATG
42
S5
TAAGTTGGCATGATTAAAGAA
21
TAATATCAAAGGCACCGCTTCTGGCACT
28
GTCGCAGAAAAACTTAAATTTGCC
24
GTTAAAGGAAAGACAGCATCTGCCTATTTAAGAGGCAGGAGGTTTA
46
AAGGCTCCAAAAGGAGCCTTTATATTTTTTCACGTGCTACAGTCACCCT
49
TAACGACATTTTTACCAGCGCCAAAGAAAGTTACCAGAACCCAAA
45
AAGGGATATTCATTACCGTAATCTATAGGCT
31
TTCGGTCCCATCGCATAGTTGCGCCGACATGCTTTCGAGGTG
42
AATATCGTTAAGAGAGCAAAGCGGATTGTGAAAAATCAGGTCTTT
45
AAATGACGCTAAATGGATTATTTACATTGGCGAATACCTGGA
42
ACCGCCACCCTCAGAACCCGTACTCTAGGGA
31
TTCGGGGTTTCTGCCAGGCCTGTGACGATCC
31
AGTACCGCATTCCACAACATGTTCAGCCTTAAGGTAAAGTAATTC
45
CCCGGTTGATAAAGCATGTCAATC
24
AGTAGGTATATGCGTTATACA
21
CTGGCATTAGGAGAATAAAATGAAGAAACGATTTTTTGAGTA
42
CGAACACCAAATAAAATAGCAGCCAAGTTTGCCTTTAGCGTCAGA
45
TGCTAAATCGGGGAGCCCCCGATTTAGAGCTAGCAGAACATT
42
CGCGCTACAGAGTAATAAAAGGGACATTCTGATAGAACTTAG
42
AAGACAAATCAGCTGCTCATTCAGTCTGACCA
32
TTTTCCAGCATCAGCGGGGCTAAAGAACCTCGTAGCACGCCA
42
ACATAAGTAGAAAAATCAAGAAGCAAAAGAAGATGTCAT
39
CAAAATCACCGGAACCAGAGCCAGATTTTGTCACAATCACAC
42
GCTGTAGTTAGAGCTTAATTG
21
TTATAAGGGTATGGAATAATTCATCAATATA
31
ATAGCGAGAGGCTATCATAACCAAATCCCAAAGAAAATTTCATCCTCAT
49
GCGAAACAAAGTGTAAAACACATGGCCTCGATTGAACCA
39
TTTAGATTCACCAGTCACACGACCGGCGCGTGCTTTCCCAGA
42
CAAGCCCAATAGGAACCACCCTCACCCGGAA
31
AGCTCTTACCGAAGCCCAATA
21
CATTTGAGATAACCCACGAAACAATG
26
AATACCCCAACATTCATCAAAAATAATTCGCGTCT
35
ACGAGCGGCGCGGTCAGGCAAGGCGATTAAGTTGGGTAAAAC
42
TAAAACCGTTAAAGAGTCTGTCCATCCAGAAACCACACAATC
42
TATTACGAATAATAAACAAATCAGATATGCGT
32
GAAGGAGCGGAATTATCATCATATATCATTTACATAGCACAA
42
CCTCGTCTTTCCACCACCGGAACCGCCTCCCTCA
34
AACAAGAGCCTAATGCAGAACGCGC
25
AGAAATCGTTAGACTACCTTTTTAAGGCGTTCTGACCTTTTTGCA
45
AGTTTATTGTCCATATAACAGTTGATTC
28
ATTTGGAAGTTTCATGCCTCAACATGTTTTA
31
S6
GAGAACAATATACAAAATCGCGCAGAGGCGATTCGACAAATCCTTTAAC
49
GTCGCGTGCCTTCGAATTGTCAAAG
25
GAACCGCCACCCTCCATATCATACC
25
CGCGCCGCCACCAGAACAGAGCCATAAAGGTGGAA
35
CAAAGCACTAGATAGCTCCATTCAGGCTGCGCAACTGTCTTG
42
GGCCAACGCGCGGGGAGGGCCCTGTGTTTGA
31
GAGGCCAAGCTTTGAATACCAAGTACGGATTACCTTTTCAAA
42
GGCGAAGCACCGTAATAACGCCAGGGTTTTCCCAGTCATGGG
42
TGAAAATCCGGTCAATAACCTAAATTTTAGCCTTT
35
TATTGAAAGGAATTGAGGTAG
21
GAGCATTTATCCTGAATCAAACGTGACTCCT
31
ATCGGTCAGATGATATTCACAAACCAAAAGA
31
GTAAAACGACGGCCCATCACCCAAATCAGCGC
32
GGCGCAGACGGTCAATCATCGAGACCTGCTCCATGTGGT
39
TTTTTGCGGATGCTCCTAAAATGTTTAGATGAATTTTGCAAAAGAAGTT
49
AAGGCCTGTTTAGTATCATGTTAGCTACCTC
31
CGAGGGTACTTTTTCATGAACGGGGTCATAATGCCGAGCCACCACC
46
AGCTTTCAGAGGTGGCGATGGCCAGCGGGAAT
32
TTGGTAGAACATTTAATTAAGCAAC
25
ACCAGACCGGATTAATTCGAGC
22
GGCAACACCAGGGTCTAATGAGTGAGCTCACAACAATAGGGT
42
ATCGATGCTGAGAGTCTACAAGGAGAGGGAACGCCAAAAGGA
42
ACCAACAAACCAAAATTAACAATTTCATTTGAATTACCGAGG
42
GCGAAAATCCCGTAAAAAAAGCCGTGGTGCTCATACCGGCGTCCG
45
GAATTCGTCTCGTCGCTGGGTCTGCAATCCATTGCAACACGG
42
AATATGCAACTACCATCATAGACCGGAACCGC
32
CCTGCGCTGGGTGGCGAGAAAGGAAGGGAAGGAGCGGGGCCG
42
CAAATTATTCATTTCAATTACCTGAGTA
28
AATTGTGTCGAAATCCGCGGCACACAACGGAGATTTGTATCA
42
TGCGTGTTCAGGTTGTGTACATCG
24
AACCGTGTCATTGCAACGGTAATATATTTTAAATGAAAGGGT
42
TGGCTTTTTACCGTAGAATGGAAAGCG
27
CGTACAGGCCCCCTAACCGTCCCCGGGTACCGAGCGTTC
39
AAGAAAGCGCTGAACCTCAAATATTCTAAAGGAAAGCGTTCA
42
TTCATCGGCATTTTCGGTCATATCAAAA
28
CCTAATTTAACAAACCCTCAATCAATATCTGATTCGCTAATC
42
AAACTCACAGGAACGGTACGCCAGTAAAGGGGGTGAGGAACC
42
AATTTCTTAAACCCGCTTAATTGTATCGTTGCGGGCGATATA
42
GAGAAGGCATCTGCAATGGGATAGGTCAAAAC
32
CCAATGTTTAAGTACGGTGTCCAAC
25
AAATCAGCTCATTTTTTAACCATTTTGTTAAAATTCGCATTA
42
S7
TTTACCAGTCCCGGCCTGCAGCCCACTACGGGCGCACCAGCT
42
CTGAATATAGAACCAAATTATTTGCACGTAAAACAACGT
39
GTAATTAATTTAGAATCTGGGAAGGGCGATCGGTGCGGCAAA
42
TAAAGCCTCCAGTACCTCATAGTTAGCG
28
AGGGAGCCGCCACGGGAACGGATAGGCGAAAGCATCAGCACTCTG
45
TGAGTGTTCCGAAAGCCCTTCACCGCCTAGGCGGTATTA
39
TTGGGCGGCTGATTTCGGCAAAATCCCT
28
CCGACTTGTTGCTAAAATTTATTTAGTTCGCGAGAGTCGTCTTTCCAGA
49
CCATAATGCCAGGCTATCAAGGCCGGAGACATCTA
35
TGACCGCGCCTTAATTTACAATATTTTTGAATGGCTATCACA
42
ACTAAAGAGCAACGTGAAAATCTCCACCCACAACTAAAGGAA
42
AGACAGCAGAAACGAAAGAGGAAATAAATCGAGGTGACAGTTAAAT
46
TTTCCATGGCACCAACCTACGTCATACA
28
TTGCGAATAATATTTACAGCGGAGTGAGGTAAAATTTTGAGG
42
ATAAAGTCTTTCCTTATCACT
21
AGGACAGATGAACGGTGTAACATAAGGGAACCGAAGAAT
39
CAAGCCGCCCAATAGCAAGTAAACAGCCATATTATTTTGCCATAAC
46
AACAACAGGAAGCACGTCCTTGCTGGTAATATCCAGAAACGC
42
ACAACGCCTGTAGCATTTACCGTATAGGAAG
31
CCGTGTGATAAATAACCTCCGGCTGATG
28
AGAATTTTAGAGGAAAACAATATTACCGCCAGCTGCTCATTT
42
AGAACTTAGCCTAATTATCCCAAGCCCCCTTATTAGCGTTTGCCA
45
CATCGAGATAACGTCAAACATAAAAGAGCAAAAGAATT
38
TTACCATTAGCAAGGCCTTGAATTAGAGCCAGCCCGACTTGAGC
44
GACAATTACGCAGAGGCATTTTCGAG
26
AATATTCATTGAATCCATGCTGGATAGCGTCCAAT
35
TTAACTCGGAATTAGAGTAAATCAATATATGTGAGTGATTCT
42
CGTGTCAAATCACCATCTAGGTAATAGATTT
31
TATCAGCAACCGCAAGAATGCCAATGAGCCTGAGGATCTATC
42
GGGATATTGACGTAGCAATAGCTAAGATAGC
31
TAAGTTTACACTGAGTTTCGT
21
ATTGCGTTGCTGTTATCCGCTCACAATTCCAAACTCACTTGCGTA
45
GCTGGCATAGCCACATTATTC
21
CGTACTATGGTAACCACTAGTCTTTAATGCGCGAACTGAATC
42
ACGGGCCGATAATCCTGAGAAGTGTTTTTATGGAGCTAACCG
42
CAAACGGAATAGGAAACCGAGGAATAAGAAATTACAAG
38
TCACAGCGTACTCCGTGGTGAAGGGATAGCTAAGAGACGAGG
42
TAACATCCAATAAATGCAAAGGTGGCATCAACATTATGAAAG
42
CAGCAGCGCCGCTTGTTTATCAGCTTCACGAAAAA
35
TAGCCCGGAATAGGTGTAAGGATAAGTGCCGTCGA
35
AAATGCGGAAACATCGGTTTTCAGGTTTAACGTCAGATTAAC
42
S8
ATTTCAACCAAAAATTCTACTAATAGTTAGTTTCATTTGGGGCGCGAGC
49
TGCTGATTGCCGTTGTCATAAACATCGGGCGG
32
GGCTAAAACTTCAGAAAAGTTTTGCGGGAGATAGAACC
38
GAGTCTGGATTTGTTATAATTACTACATACACCAC
35
ATTGTTATCTGAGAAGAAACCAGGCAAAGCGCCATTCGTAGA
42
CGGAATAGAAAGGAATGCCTTGCTAAACAACTTTCAAC
38
CTAGTCAGTTGGCAAATCAACAGTCTTTAGGTAGATAACAAA
42
AGTCGCCTGATACTTGCATAACAGAATACGTGGCACAGCTGA
42
CACGGCAACAATCCTGATATACTT
24
CCTCATCACCCCAGCAGGCCTCTTCGCTATTACGCCAGTGCC
42
TGAGCAAATTTATACAGGAATAACATCACTTGCCTGAGTCTT
42
AATAGAAAAAAATAAACGTCTGAGAGGAATATAAGAGCAACACTATGAT
49
ATTACGAGATAAATGCCAGCTTTGAGGGGACGACGACAG
39
GCTGGTCTGGTCAGGAGCCGGAATCCGCCGTGAACAGTGCCA
42
CTTGTAGAACGTCAGCGGCTGATTGCAGAGTTTTTCGACGTT
42
ACATAAAGCCCTTACACTGGTCGGGTTAAATTTGT
35
TGCCATCCCACGCAGGCAGTTCCTCATTGCCGTTTTAAACGA
42
GCCAGCAGTTGGGCGCAAATCAGGTTTCTTGCCCTGCGTGGT
42
TACGGCTGGAGGTGCGCACTCGTCACTGTTTGCTCCCGGCAA
42
GAGAGATAGACTTTACGGCATCAGA
25
ATTAGCGGGGTTTTGCTCAGTACCAGGCTGACAACAAGCTG
41
5'-Biotin
TGCCCGTATAAACAGTGTGCCTTCTGGTAA
30
5'-Biotin
AGAAAACGAGAATGACCATAAATCTACGCCCCTCAAATGCTTTA
44
5'-Biotin
ATAACTATATGTAAATGCTTAGGATATAAT
30
5'-Biotin
AGGAATCATTACCGCGTTTTTATAAGTACC
30
5'-Biotin
GATTAGAGAGTACCTTAACTCCAACAGG
28
5'-Biotin
CCTTAAATCAAGATTAGCGGGAGGCTCAAC
30
5'-Biotin
GCATGTAGAAACCAATCCATCCTAGTCCTG
30
5'-Biotin
TGCATTAATGAGCGGTCCACGCTCACTGCGCCACGTGCCAGCAAAAAAAAAAAAAAAAAAAA
62
NP binding
AGCGCAGCTCCAACCGTAATCATGGTCACGGGAAACCTAAAAAAAAAAAAAAAAAAAA
58
NP binding
GCGTCCACTATTCCTGTGTGAAATGCTCACTGCCAAAAAAAAAAAAAAAAAAAA
54
NP binding
TGGTGGTTGTTCCAGTTTGGAACAAAAAAAAAAAAAAAAAAAAA
44
NP binding
GGATGTGGTTTGCCCCAGCAGAAAAAAAAAAAAAAAAAAAA
41
NP binding
CGCTTTCCAGTTAGCTGTTTAAAGAACGTAAAAAAAAAAAAAAAAAAAA
49
NP binding
AGAGAAAATCCAGAGAGTTGCAGCAAATC
29
ATTO647N-3’
Table S2. Detailed folding program for the DNA origami pillar.
S9
temp [°C] time [s] temp [°C] time [s] temp [°C] time [s] temp [°C] time [s]
65 120 52 5400 39 1800 26 120
64 180 51 5400 38 900 25 120
63 180 50 5400 37 480 20 ∞
62 180 49 5400 36 480
61 180 48 5400 35 480
60 900 47 5400 34 480
59 900 46 5400 33 480
58 1800 45 5400 32 480
57 2700 44 4500 31 480
56 3600 43 3600 30 480
55 4500 42 2700 29 120
54 5400 41 1800 28 120
53 5400 40 1800 27 120
References 1. Douglas, S. M.; Marblestone, A. H.; Teerapittayanon, S.; Vazquez, A.; Church, G. M.; Shih, W. M. Nucleic Acids Research 2009, 37, (15), 5001-5006. 2. Mirkin, C.; Letsinger, R.; Mucic, R.; Storhoff, J. Nature 1996, 382, 607−609.
S10
DNA Origami Nanoantennas with over 5000fold Fluorescence Enhancement and Single-Molecule Detection at 25 µM Anastasiya Puchkova, Carolin Vietz, Bettina Wünsch, Enrico Pibiri, Maria Sanz Paz, Guillermo P. Acuna* and Philip Tinnefeld Institute for Physical & Theoretical Chemistry, and Braunschweig Integrated Centre of Systems Biology (BRICS), and Laboratory for Emerging Nanometrology (LENA), Braunschweig University of Technology, 38106 Braunschweig, Germany. *Correspondence to: [email protected]
Sample preparation of DNA origami pillar DNA origami pillar was designed using the CaDNAno software (http://cadnano.org/) (1). For the folding of this structure 10 nM of 8064 nt long single stranded DNA scaffold (M13mp18) and 100 nM of each staple strand (see table S1, purchased from Eurofins Genomics) were mixed in a TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0) containing 14 mM MgCl2. The folding mixture was then annealed following the program presented in table S2. After folding the sample was purified from excess staple strands by filtering in Amicon columns (Amicon Ultra – 0.5 ml, Ultracel®- 100K Membrane, Millipore) with TAE buffer (40 mM Tris, 2 mM EDTA) containing 12 mM MgCl2. The sample was centrifuged 3 times at 16 krcf speed for 5 min at 20°C with buffer replacement after each step. To remove purified origami structures the filter column was flipped into a new tube and centrifuged for 3 min at 1 krcf speed.
S1
Functionalization of gold nanoparticles Gold nanoparticles of 100 nm diameter (BBI solutions) were functionalized with 20T DNAoligonucleotides containing a thiol modification on the 3’ end (Ella Biotech GmbH) following the procedure adapted from Mirkin (2) with minor changes. The nanoparticle oligonucleotide mixture was stirred at 40 °C during the salting procedure. By stepwise adding 1xPBS buffer containing 3.3 M NaCl a final concentration of 750 mM NaCl was reached. For purification from free oligonucleotides the nanoparticle solution was spinned down, the supernatant was pipetted off and the nanoparticle pellet was diluted in 1xPBS containing 10 mM NaCl, 2.11 mM P8709, 2.89 mM P8584, 0.01 % Tween20 and 1 mM EDTA-S. This purification step was repeated 7 times.
Figure S1. “Immobilized reference” refers to the quenching of the fluorophore bound to the DNA origami and immobilized on the surface of a coverslip. Therefore the fluorescence intensity of single ATTO647N was determined at different concentrations of NiCl2 as indicated and referred to the reference in the absence of quencher.
S2
Figure S2. Fluorescence quenching of ATTO647N by NiCl2 on the ensemble level measured in a spectrometer. ATTO647N 3’-labelled oligonucleotides with the following sequence were used: GCG CAT CAT CTG CCA GTT TTC ACC AGG CGT GAG CAA GTC TTG
S3
Figure S3. Photon count rate histogram for the dimer nanoantennas with an ATTO655 fluorophore (red) and an ATTO647N with a NiCl2 concentration of 0.5 mM (black). The enhancement is calculated based on the comparison with a reference that consist of an immobilized DNA origami pillar with the same fluorophore and quencher conditions in the absence of any plasmonic interaction.
S4
Table S1. List of unmodified and modified staples from the 5’ to the 3’ end for the DNA origami pillar. Sequence (5’ to 3’)
Length
Modification
TTAGTTTGAGTGCCCGAGAAATAAAGAAATTGCGTAGAGATA
42
TATGACTTTATACATTTTTTTTTAATGGAAACAGTACACCGT
42
AATAAAACGAACTATGACCCCACCAAGC
28
CTCATCGGGATTGAGTGAGCGAGTAACAACCCGTC
35
CCCAGCTACAATGACAGCATTTGAGGCAAGTTGAGAAATGAA
42
TCATACATTTAATACCGATAGCCCTAAAACATCGAACGTAAC
42
ACGCGGTCCGTTTTTGGGTAAGTGA
25
CTTACGGAACAGTCAGGACGTTGGGAAGAAA
31
GGAACCATACAGGCAAGGCAAATCAAAAAGACGTAGTAGCAT
42
CTAAATCGGTCAGAATTAGCAAAATTAAGCAATAAAATAATA
42
TTTAGCGATACCAACGCGTTA
21
AAGAAAGCTTGATACCGCCACGCATACAGACCAGGCGCTGAC
42
CCCCGCTAGGGCAACAGCTGGCGAAAGGGGGATGTGCTTATT
42
GCCCGAGTACGAGCCGGAAGC
21
CATTTCGCAAATGTCATCTGCGAACGAGAGATTCACAATGCC
42
TATTTAAATTGCAGGAAGATTG
22
AAAGATTACAGAACGGGAGAAGGAAACGTCACCAATGAAACCA
43
ACGTAAGAATTCGTTCTTAGAAGAACTCAAACTATCGGATAA
42
GCGAATCAGTGAGGCCACCGAGTAGTAGCAACTGAGAGTTGA
42
TCGTGCCGGAGTCAATAGTGAATTTGCAGAT
31
TAGCCTCAGAGCATACCCTGT
21
AGCAACAAAGTCAGAAATAATATCCAATAATCGGCTCAGGGA
42
AGGCTTGCGAGACTCCTCAAGAGAAAAGTATTCGGAAC
38
ACCTGACGGGGAAAGCCGGCGAACCAAGTGTCTGCGCGTTGC
42
CCGTAATCAGTAGCGACAGAATCTAATTATTCATTAAAAAGG
42
GAACTGGCTCATTACAACTTTAATCATTCTTGAGATTACTTA
42
GAGTTAAAAGGGTAATTGAGCGCTAATATCAGAGGAACTGAACACC
46
AGTTTCCAACATTATTACATTATAC
25
ACGCGAGAGAAGGCCATGTAATTTAGGCCAGGCTTAATTGAGAATCGC
48
ACTAATGCCACTACGAATAAA
21
CTGTATGGGATTACCGTTAGTATCA
25
TAGCCAGCTTTCATCCAAAAATAAACGT
28
CCTCGTTTACCAGAAACCAAA
21
TTAGCCCTGACGAGAAACACCAGAAATTGGGGTGAATTATTTTAA
45
TGAGTAAAGGATAAGTTTAGCTATATCATAGACCATTAGATA
42
ATTTCCTGATTATCAGATGATGGCTTTAAAAAGACGCTAAAA
42
CCAGCCTCCGATCCTCATGCCGGA
24
ATGAAGGGTAAAGTTCACGGTGCGGCCATGCCGGTCGCCATG
42
S5
TAAGTTGGCATGATTAAAGAA
21
TAATATCAAAGGCACCGCTTCTGGCACT
28
GTCGCAGAAAAACTTAAATTTGCC
24
GTTAAAGGAAAGACAGCATCTGCCTATTTAAGAGGCAGGAGGTTTA
46
AAGGCTCCAAAAGGAGCCTTTATATTTTTTCACGTGCTACAGTCACCCT
49
TAACGACATTTTTACCAGCGCCAAAGAAAGTTACCAGAACCCAAA
45
AAGGGATATTCATTACCGTAATCTATAGGCT
31
TTCGGTCCCATCGCATAGTTGCGCCGACATGCTTTCGAGGTG
42
AATATCGTTAAGAGAGCAAAGCGGATTGTGAAAAATCAGGTCTTT
45
AAATGACGCTAAATGGATTATTTACATTGGCGAATACCTGGA
42
ACCGCCACCCTCAGAACCCGTACTCTAGGGA
31
TTCGGGGTTTCTGCCAGGCCTGTGACGATCC
31
AGTACCGCATTCCACAACATGTTCAGCCTTAAGGTAAAGTAATTC
45
CCCGGTTGATAAAGCATGTCAATC
24
AGTAGGTATATGCGTTATACA
21
CTGGCATTAGGAGAATAAAATGAAGAAACGATTTTTTGAGTA
42
CGAACACCAAATAAAATAGCAGCCAAGTTTGCCTTTAGCGTCAGA
45
TGCTAAATCGGGGAGCCCCCGATTTAGAGCTAGCAGAACATT
42
CGCGCTACAGAGTAATAAAAGGGACATTCTGATAGAACTTAG
42
AAGACAAATCAGCTGCTCATTCAGTCTGACCA
32
TTTTCCAGCATCAGCGGGGCTAAAGAACCTCGTAGCACGCCA
42
ACATAAGTAGAAAAATCAAGAAGCAAAAGAAGATGTCAT
39
CAAAATCACCGGAACCAGAGCCAGATTTTGTCACAATCACAC
42
GCTGTAGTTAGAGCTTAATTG
21
TTATAAGGGTATGGAATAATTCATCAATATA
31
ATAGCGAGAGGCTATCATAACCAAATCCCAAAGAAAATTTCATCCTCAT
49
GCGAAACAAAGTGTAAAACACATGGCCTCGATTGAACCA
39
TTTAGATTCACCAGTCACACGACCGGCGCGTGCTTTCCCAGA
42
CAAGCCCAATAGGAACCACCCTCACCCGGAA
31
AGCTCTTACCGAAGCCCAATA
21
CATTTGAGATAACCCACGAAACAATG
26
AATACCCCAACATTCATCAAAAATAATTCGCGTCT
35
ACGAGCGGCGCGGTCAGGCAAGGCGATTAAGTTGGGTAAAAC
42
TAAAACCGTTAAAGAGTCTGTCCATCCAGAAACCACACAATC
42
TATTACGAATAATAAACAAATCAGATATGCGT
32
GAAGGAGCGGAATTATCATCATATATCATTTACATAGCACAA
42
CCTCGTCTTTCCACCACCGGAACCGCCTCCCTCA
34
AACAAGAGCCTAATGCAGAACGCGC
25
AGAAATCGTTAGACTACCTTTTTAAGGCGTTCTGACCTTTTTGCA
45
AGTTTATTGTCCATATAACAGTTGATTC
28
ATTTGGAAGTTTCATGCCTCAACATGTTTTA
31
S6
GAGAACAATATACAAAATCGCGCAGAGGCGATTCGACAAATCCTTTAAC
49
GTCGCGTGCCTTCGAATTGTCAAAG
25
GAACCGCCACCCTCCATATCATACC
25
CGCGCCGCCACCAGAACAGAGCCATAAAGGTGGAA
35
CAAAGCACTAGATAGCTCCATTCAGGCTGCGCAACTGTCTTG
42
GGCCAACGCGCGGGGAGGGCCCTGTGTTTGA
31
GAGGCCAAGCTTTGAATACCAAGTACGGATTACCTTTTCAAA
42
GGCGAAGCACCGTAATAACGCCAGGGTTTTCCCAGTCATGGG
42
TGAAAATCCGGTCAATAACCTAAATTTTAGCCTTT
35
TATTGAAAGGAATTGAGGTAG
21
GAGCATTTATCCTGAATCAAACGTGACTCCT
31
ATCGGTCAGATGATATTCACAAACCAAAAGA
31
GTAAAACGACGGCCCATCACCCAAATCAGCGC
32
GGCGCAGACGGTCAATCATCGAGACCTGCTCCATGTGGT
39
TTTTTGCGGATGCTCCTAAAATGTTTAGATGAATTTTGCAAAAGAAGTT
49
AAGGCCTGTTTAGTATCATGTTAGCTACCTC
31
CGAGGGTACTTTTTCATGAACGGGGTCATAATGCCGAGCCACCACC
46
AGCTTTCAGAGGTGGCGATGGCCAGCGGGAAT
32
TTGGTAGAACATTTAATTAAGCAAC
25
ACCAGACCGGATTAATTCGAGC
22
GGCAACACCAGGGTCTAATGAGTGAGCTCACAACAATAGGGT
42
ATCGATGCTGAGAGTCTACAAGGAGAGGGAACGCCAAAAGGA
42
ACCAACAAACCAAAATTAACAATTTCATTTGAATTACCGAGG
42
GCGAAAATCCCGTAAAAAAAGCCGTGGTGCTCATACCGGCGTCCG
45
GAATTCGTCTCGTCGCTGGGTCTGCAATCCATTGCAACACGG
42
AATATGCAACTACCATCATAGACCGGAACCGC
32
CCTGCGCTGGGTGGCGAGAAAGGAAGGGAAGGAGCGGGGCCG
42
CAAATTATTCATTTCAATTACCTGAGTA
28
AATTGTGTCGAAATCCGCGGCACACAACGGAGATTTGTATCA
42
TGCGTGTTCAGGTTGTGTACATCG
24
AACCGTGTCATTGCAACGGTAATATATTTTAAATGAAAGGGT
42
TGGCTTTTTACCGTAGAATGGAAAGCG
27
CGTACAGGCCCCCTAACCGTCCCCGGGTACCGAGCGTTC
39
AAGAAAGCGCTGAACCTCAAATATTCTAAAGGAAAGCGTTCA
42
TTCATCGGCATTTTCGGTCATATCAAAA
28
CCTAATTTAACAAACCCTCAATCAATATCTGATTCGCTAATC
42
AAACTCACAGGAACGGTACGCCAGTAAAGGGGGTGAGGAACC
42
AATTTCTTAAACCCGCTTAATTGTATCGTTGCGGGCGATATA
42
GAGAAGGCATCTGCAATGGGATAGGTCAAAAC
32
CCAATGTTTAAGTACGGTGTCCAAC
25
AAATCAGCTCATTTTTTAACCATTTTGTTAAAATTCGCATTA
42
S7
TTTACCAGTCCCGGCCTGCAGCCCACTACGGGCGCACCAGCT
42
CTGAATATAGAACCAAATTATTTGCACGTAAAACAACGT
39
GTAATTAATTTAGAATCTGGGAAGGGCGATCGGTGCGGCAAA
42
TAAAGCCTCCAGTACCTCATAGTTAGCG
28
AGGGAGCCGCCACGGGAACGGATAGGCGAAAGCATCAGCACTCTG
45
TGAGTGTTCCGAAAGCCCTTCACCGCCTAGGCGGTATTA
39
TTGGGCGGCTGATTTCGGCAAAATCCCT
28
CCGACTTGTTGCTAAAATTTATTTAGTTCGCGAGAGTCGTCTTTCCAGA
49
CCATAATGCCAGGCTATCAAGGCCGGAGACATCTA
35
TGACCGCGCCTTAATTTACAATATTTTTGAATGGCTATCACA
42
ACTAAAGAGCAACGTGAAAATCTCCACCCACAACTAAAGGAA
42
AGACAGCAGAAACGAAAGAGGAAATAAATCGAGGTGACAGTTAAAT
46
TTTCCATGGCACCAACCTACGTCATACA
28
TTGCGAATAATATTTACAGCGGAGTGAGGTAAAATTTTGAGG
42
ATAAAGTCTTTCCTTATCACT
21
AGGACAGATGAACGGTGTAACATAAGGGAACCGAAGAAT
39
CAAGCCGCCCAATAGCAAGTAAACAGCCATATTATTTTGCCATAAC
46
AACAACAGGAAGCACGTCCTTGCTGGTAATATCCAGAAACGC
42
ACAACGCCTGTAGCATTTACCGTATAGGAAG
31
CCGTGTGATAAATAACCTCCGGCTGATG
28
AGAATTTTAGAGGAAAACAATATTACCGCCAGCTGCTCATTT
42
AGAACTTAGCCTAATTATCCCAAGCCCCCTTATTAGCGTTTGCCA
45
CATCGAGATAACGTCAAACATAAAAGAGCAAAAGAATT
38
TTACCATTAGCAAGGCCTTGAATTAGAGCCAGCCCGACTTGAGC
44
GACAATTACGCAGAGGCATTTTCGAG
26
AATATTCATTGAATCCATGCTGGATAGCGTCCAAT
35
TTAACTCGGAATTAGAGTAAATCAATATATGTGAGTGATTCT
42
CGTGTCAAATCACCATCTAGGTAATAGATTT
31
TATCAGCAACCGCAAGAATGCCAATGAGCCTGAGGATCTATC
42
GGGATATTGACGTAGCAATAGCTAAGATAGC
31
TAAGTTTACACTGAGTTTCGT
21
ATTGCGTTGCTGTTATCCGCTCACAATTCCAAACTCACTTGCGTA
45
GCTGGCATAGCCACATTATTC
21
CGTACTATGGTAACCACTAGTCTTTAATGCGCGAACTGAATC
42
ACGGGCCGATAATCCTGAGAAGTGTTTTTATGGAGCTAACCG
42
CAAACGGAATAGGAAACCGAGGAATAAGAAATTACAAG
38
TCACAGCGTACTCCGTGGTGAAGGGATAGCTAAGAGACGAGG
42
TAACATCCAATAAATGCAAAGGTGGCATCAACATTATGAAAG
42
CAGCAGCGCCGCTTGTTTATCAGCTTCACGAAAAA
35
TAGCCCGGAATAGGTGTAAGGATAAGTGCCGTCGA
35
AAATGCGGAAACATCGGTTTTCAGGTTTAACGTCAGATTAAC
42
S8
ATTTCAACCAAAAATTCTACTAATAGTTAGTTTCATTTGGGGCGCGAGC
49
TGCTGATTGCCGTTGTCATAAACATCGGGCGG
32
GGCTAAAACTTCAGAAAAGTTTTGCGGGAGATAGAACC
38
GAGTCTGGATTTGTTATAATTACTACATACACCAC
35
ATTGTTATCTGAGAAGAAACCAGGCAAAGCGCCATTCGTAGA
42
CGGAATAGAAAGGAATGCCTTGCTAAACAACTTTCAAC
38
CTAGTCAGTTGGCAAATCAACAGTCTTTAGGTAGATAACAAA
42
AGTCGCCTGATACTTGCATAACAGAATACGTGGCACAGCTGA
42
CACGGCAACAATCCTGATATACTT
24
CCTCATCACCCCAGCAGGCCTCTTCGCTATTACGCCAGTGCC
42
TGAGCAAATTTATACAGGAATAACATCACTTGCCTGAGTCTT
42
AATAGAAAAAAATAAACGTCTGAGAGGAATATAAGAGCAACACTATGAT
49
ATTACGAGATAAATGCCAGCTTTGAGGGGACGACGACAG
39
GCTGGTCTGGTCAGGAGCCGGAATCCGCCGTGAACAGTGCCA
42
CTTGTAGAACGTCAGCGGCTGATTGCAGAGTTTTTCGACGTT
42
ACATAAAGCCCTTACACTGGTCGGGTTAAATTTGT
35
TGCCATCCCACGCAGGCAGTTCCTCATTGCCGTTTTAAACGA
42
GCCAGCAGTTGGGCGCAAATCAGGTTTCTTGCCCTGCGTGGT
42
TACGGCTGGAGGTGCGCACTCGTCACTGTTTGCTCCCGGCAA
42
GAGAGATAGACTTTACGGCATCAGA
25
ATTAGCGGGGTTTTGCTCAGTACCAGGCTGACAACAAGCTG
41
5'-Biotin
TGCCCGTATAAACAGTGTGCCTTCTGGTAA
30
5'-Biotin
AGAAAACGAGAATGACCATAAATCTACGCCCCTCAAATGCTTTA
44
5'-Biotin
ATAACTATATGTAAATGCTTAGGATATAAT
30
5'-Biotin
AGGAATCATTACCGCGTTTTTATAAGTACC
30
5'-Biotin
GATTAGAGAGTACCTTAACTCCAACAGG
28
5'-Biotin
CCTTAAATCAAGATTAGCGGGAGGCTCAAC
30
5'-Biotin
GCATGTAGAAACCAATCCATCCTAGTCCTG
30
5'-Biotin
TGCATTAATGAGCGGTCCACGCTCACTGCGCCACGTGCCAGCAAAAAAAAAAAAAAAAAAAA
62
NP binding
AGCGCAGCTCCAACCGTAATCATGGTCACGGGAAACCTAAAAAAAAAAAAAAAAAAAA
58
NP binding
GCGTCCACTATTCCTGTGTGAAATGCTCACTGCCAAAAAAAAAAAAAAAAAAAA
54
NP binding
TGGTGGTTGTTCCAGTTTGGAACAAAAAAAAAAAAAAAAAAAAA
44
NP binding
GGATGTGGTTTGCCCCAGCAGAAAAAAAAAAAAAAAAAAAA
41
NP binding
CGCTTTCCAGTTAGCTGTTTAAAGAACGTAAAAAAAAAAAAAAAAAAAA
49
NP binding
AGAGAAAATCCAGAGAGTTGCAGCAAATC
29
ATTO647N-3’
Table S2. Detailed folding program for the DNA origami pillar.
S9
temp [°C] time [s] temp [°C] time [s] temp [°C] time [s] temp [°C] time [s]
65 120 52 5400 39 1800 26 120
64 180 51 5400 38 900 25 120
63 180 50 5400 37 480 20 ∞
62 180 49 5400 36 480
61 180 48 5400 35 480
60 900 47 5400 34 480
59 900 46 5400 33 480
58 1800 45 5400 32 480
57 2700 44 4500 31 480
56 3600 43 3600 30 480
55 4500 42 2700 29 120
54 5400 41 1800 28 120
53 5400 40 1800 27 120
References 1. Douglas, S. M.; Marblestone, A. H.; Teerapittayanon, S.; Vazquez, A.; Church, G. M.; Shih, W. M. Nucleic Acids Research 2009, 37, (15), 5001-5006. 2. Mirkin, C.; Letsinger, R.; Mucic, R.; Storhoff, J. Nature 1996, 382, 607−609.
S10
