Electronic Supplementary Material (ESI) for ChemComm.
This journal is © The Royal Society of Chemistry 2014
Supporting Information
Lock-and-key mechanism for the controllable fabrication of DNA
origami structures
Arivazhagan Rajendran,ab Masayuki Endo,*cd Kumi Hidaka,a Naohiko Shimada,e Atsushi
Maruyama,e and Hiroshi Sugiyama*acd
a
Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto
606-8502, Japan. Email: [email protected]
b
Current addresses: Faculty of Medicine and Life Science Center of TARA, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba-shi, Ibaraki-ken 305-8577, Japan
c
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku,
Kyoto 606-8501, Japan. Email: [email protected]
d
CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
e
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of
Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan
EXPERIMENTAL SECTION
Chemicals and Reagents. Tris-HCl, EDTA and MgCl2 were purchased from Nacalai Tesque, Inc.
(Kyoto, Japan). Polyvinyl sulphonic acid was purchased from Sigma-Aldrich Co. Single-stranded
M13mp18 ssDNA was obtained from New England Biolabs, Inc. (Ipswich, MA, catalog no.
N4040S). The staple strands for the fabrication of the DNA origami were received from Sigma
Genosys (Hokkaido, Japan). The gel-filtration column and sephacryl S-500 were purchased from
Bio-Rad Laboratories, Inc. (Hercules, CA) and GE Healthcare UK Ltd. (Buckinghamshire, UK),
respectively. Water was deionized (≥18.0 MΩ cm specific resistance at 25 °C) by a Milli-Q system
(Millipore Corp., Bedford, MA). Cationic comb-type copolymers were prepared and purified as we
have described previously (Maruyama et al. Bioconjugate Chem., 1997, 8, 3-6; and Maruyama et al.
Bioconjugate Chem., 1998, 9, 292-299).
Design of the DNA Origami. In the present study, we used the jigsaw-shaped DNA origami (JP-1)
that we have designed previously. It is a 24-helix tile with the size of ~100 nm × 80 nm. The
detailed design of JP-1 and the sequences of the staple strands are given in Supporting Information
of (Rajendran et al. ACS Nano, 2011, 5, 665-671). The outline of the JP-1 origami structure is also
given in Figure S1.
Lock-and-Key Mechanism for the Preparation of Origami. The origami structure was prepared
by following the procedure originally described by Rothemund (Rothemund, P. W. K. Nature 2006,
440, 297). For a step-by-step procedure for the preparation and purification of “Rothemund’s
scaffolded DNA origami structures”, see our recent protocol (Rajendran et al. Curr. Protoc. Nucleic
Acid Chem. 2012, 48, 12.9.1). The lock-and-key mechanism for the controllable fabrication of
origami adopts the following procedures:
a) Lock M13mp18 and release using PVS:
i) M13mp18 ssDNA (final concentration of 10 nM) was mixed with 1x Tris-HCl buffer
(final concentration: 20 mM Tris-HCl, pH 7.6, 10 mM MgCl2 and 1 mM EDTA) and CCC
(with desired N/P ratio)
ii) The above solution mixture was incubated at room temperature for 30 min, so that the
CCC can sufficiently bind to the M13mp18
iii) Staple DNA strands (4 equiv., 40 nM) and PVS (5 times higher concentration than that
of the CCC) was added to the solution
iv) The solution was annealed from 85ºC to 15ºC (-1ºC/min)
v) The solution was purified using sephacryl S-500 gel filtration column (prepared in 1x
Tris-HCl buffer) and imaged using high-speed atomic force microscopy (HS-AFM)
b) Lock staple strands and release using PVS:
i) Staple strands (final concentration of 40 nM) was mixed with 1x Tris-HCl buffer and
CCC (with desired N/P ratio)
ii) The above solution mixture was incubated at room temperature for 30 min, so that the
CCC can sufficiently bind to the staple strands
iii) M13mp18 (final concentration of 10 nM) and PVS (5 times higher concentration than
that of the CCC) was added to the solution
iv) The solution was annealed from 85ºC to 15ºC (-1ºC/min)
v) The solution was purified using sephacryl S-500 gel filtration column and imaged using
HS-AFM
c) Lock both M13mp18 and staple strands, and release using PVS:
i) M13mp18 (final concentration of 10 nM) was mixed with staple strands (4 equiv., 40 nM),
1x Tris-HCl buffer and CCC (with desired N/P ratio)
ii) The above solution mixture was incubated at room temperature for 30 min, so that the
CCC can sufficiently bind to M13mp18 and staple strands
iii) PVS (5 times higher concentration than that of the CCC) was added to the solution
iv) The solution was annealed from 85ºC to 15ºC (-1ºC/min)
v) The solution was purified using sephacryl S-500 gel filtration column and imaged using
HS-AFM
Control experiments:
For all three methods mentioned above, we have also performed control experiments in
which all the experimental steps are same as above while no PVS was added.
Experiments in the presence of phosphate buffer:
Instead of Tris-HCl, phosphate buffer (with the mentioned concentrations) was used in all
the three methods mentioned above. In this case no PVS was added.
Experiments in the presence of mononucleotide triphosphates (NTPs):
In all the three methods mentioned above, NTPs (with the mentioned concentrations) were
used instead of PVS.
N/P ratio is defined as:
N/P ratio = {[amino groups]copolymer/[phosphate groups]DNA}
where, DNA = M13mp18 + staple strands
AFM Imaging. AFM images were recorded using a fast-scanning AFM system (Nano Live Vision,
RIBM Co. Ltd., Tsukuba, Japan) with a silicon nitride cantilever (resonant frequency 1.0-2.0 MHz,
spring constant 0.1-0.3 N/m, EBDTip radius <15 nm, Olympus BL-AC10EGS-A2). The sample (2
µL) was adsorbed onto a freshly cleaved mica plate (φ 1.5 mm, RIBM Co. Ltd., Tsukuba, Japan) for
5 min at room temperature and then the surface was gently washed 3-5 times using 1x Tris-HCl
buffer solution. Scanning was performed by tapping mode in the same buffer solution. All images
reported here were recorded with an image acquisition speed of 0.2 frame/s.
1
2
3
ACAGTAAC AGCCGCGT TGATAGCC ATAGTTCG ACAAATTC TTTAAGTG
4
5
6
7
9
8
10
11
12
13
14
15
16
17
18
GAGCTTTC GTTCGACT ATTTGGCT ATGTTAAT TTCCGAGG AAAACCTC GGAAAAAA AACCTCTA AAAGTTGC ACTTTTTT
TGTCATTG TCGGCGCA ACTATCGG TATCAAGC TGTTTAAG AAATTCAC - CTCGAAAG CAAGCTGA TAAACCGA TACAATTA AAGGCTCC TTTTGGAG CCTTTTTT TTGGAGAT TTTCAACG TGAAAAAA
GCAAGCCT CAGCGACC GAATATAT CGGTTATG CGTGGGCG ATGGTTGT
AAAGCGAC GACTCCCA CTGCTAGG GCGTTTTC GCCGGAAA TTGAGGGA
ATGTCTTT TAAGTAAA TGATTGCA GACCTTTC TGCTGTTT TGAAATCT AGCAATGC GATTGATA CTCCCGAC AGACACCT
TTTCGCTG CTGAGGGT GACGATCC CGCAAAAG CGGCCTTT AACTCCCT
TACAGAAA ATTCATTT ACTAACGT CTGGAAAG ACGACAAA ACTTTAGA TCGTTACG CTAACTAT GAGGGCTG TCTGTGGA
TCAGGAGT TTCGGAGA CATCGGCA ACGATGGG AGCAAGGC TACGACAG
TACGATGT CCGCAACA TCAAACAT GACCACTG CTTTGAGT CACAATGC CATGTACC CAAGGATA ACCCGAAC GATAGGGA
AGTCCTCA AAGCCTCT GTAGCCGT TGCTACCC TCGTTCCG ATGCTGTC
ATGCTACA GGCGTTGT AGTTTGTA CTGGTGAC GAAACTCA GTGTTACG GTACATGG GTTCCTAT TGGGCTTG CTATCCCT
AATGCATA AAATGGGC AAATTACC TTTGAAGG AGTACTTT TTCAGAAA
CTTTTACT CCCACCAC CGAGACTC CCACCGCC AAGACTCC CACCGCCA AGACTCCC ACCGCCAT GATTTGGA GGACTCAT GCCACTAT GTGGATAA
GGCCCGAT ATGAATAT AGTTGGGA GAGCTGCC GTGAATAG GCGGACCA TGACTCGT TTTGGGGC GATTAGGA TTAGGAAG AGAACTCC TCAGAGTC GGAGAATT ATGAAAGT ACAAAGTC TTATTATC CAAGGCTT TATCCGTC
TTACGTAT TTTACCCG TTTAATGG AAACTTCC TCATGAAA AAGTCTTT
GAAAATGA GGGTGGTG GCTCTGAG GGTGGCGG TTCTGAGG GTGGCGGT TCTGAGGG TGGCGGTA CTAAACCT CCTGAGTA CGGTGATA CACCTATT
CCGGGCTA TACTTATA TCAACCCT CTCGACGG CACTTATC CGCCTGGT ACTGAGCA AAACCCCG CTAATCCT AATCCTTC TCTTGAGG AGTCTCAG CCTCTTAA TACTTTCA TGTTTCAG AATAATAG GTTCCGAA ATAGGCAG
CTAAAGCT GTGTTAAA TAGTCCGC TACTATGT TTAGAGGC AACATGAA ACAAAGCG CGAACCAT ATTAGCGA CCCCCAGT TTCTACTC ACAAAATC ACATAAGA AAACGGAG AAAGCAAA ATCCAACC ACGGAAGC ATCACCGT
CCCCGTAA TTGACAAA TATGCCCG TGACAATG AGTTCCGT GACTGGGG CAATTTTG AATAATGG TCATGTGA GGACATAG TAGTTTTC GGTACATA CTGCGAAT GACCTTGC CATTTAAG TCTCTGAC GCGAAAGG TAAGACCG
GATTTCGA CACAATTT ATCAGGCG ATGATACA AATCTCCG TTGTACTT TGTTTCGC GCTTGGTA TAATCGCT GGGGGTCA AAGATGAG TGTTTTAG TGTATTCT TTTGCCTC TTTCGTTT TAGGTTGG TGCCTTCG TAGTGGCA
TCTAATGA GAACTACT TCCAGTCG GTCGGATA CGCGGACC AGACATGT GGCAAGTA GACAGGAG AAAGTTTC AACCAGTC AAGCCAAG GGAATACT AACTGGCA GACGCGGA GCAAGGCC GATTCATT GTACCTCG TCCAGCGC
AAATTACT CCTAAATA AACAAACA CTTATAGT TCCGGTTA GCAGACTG GACGGAGT TGGAGGAC AGTTACGA CCGCCGCC GAGACCAC CACCAAGA CCACCGCC GAGACTCC CACCACCG AGACTCCC ACCGCCAA GACTCCCA
AGATTACT CTTGATGA AGGTCAGC CAGCCTAT GCGCCTGG TCTGTACA CCGTTCAT CTGTCCTC TTTCAAAG TTGGTCAG TTCGGTTC CCTTATGA TTGACCGT CTGCGCCT CGTTCCGG CTAAGTAA CATGGAGC AGGTCGCG
TTTAATGA GGATTTAT TTGTTTGT GAATATCA AGGCCAAT CGTCTGAC CTGCCTCA ACCTCCTG TCAATGCT GGCGGCGG CTCTGGTG GTGGTTCT GGTGGCGG CTCTGAGG GTGGTGGC TCTGAGGG TGGCGGTT CTGAGGGT
TTAAGTGT TACTAATT TCAACTTT AATTTGGT AGAGTTCG GGTTAAAT GATGAGCA AGACCACA AAGAGCAG TCCCGTTC GGAATAAG TGACTTAC TCGTCGAA ACAATGCA ACTAAACC CATTACTT ATAGGCCA AGAACAGT
CCGCCGAG ACTCCCTC CGCCAAGG CCACCACC GAGACCAA GGCCACTA AAACTAAT ACTTTTCT ACCGTTTG CGATTATT CCCCCGAT ACTGGCTT TTACGGCT ACTTTTGC GCGATGTC AGACTGCG ATTTCCGT TTGAACTA
AATTCACA ATGATTAA AGTTGAAA TTAAACCA TCTCAAGC CCAATTTA CTACTCGT TCTGGTGT TTCTCGTC AGGGCAAG CCTTATTC ACTGAATG AGCAGCTT TGTTACGT TGATTTGG GTAATGAA TATCCGGT TCTTGTCA
GGCGGCTC TGAGGGAG GCGGTTCC GGTGGTGG CTCTGGTT CCGGTGAT TTTGATTA TGAAAAGA TGGCAAAC GCTAATAA GGGGGCTA TGACCGAA AATGCCGA TGAAAACG CGCTACAG TCTGACGC TAAAGGCA AACTTGAT
AGACAGCG ATGACTAA TGCCACGA CGATAGCT ACCAAAGT AACCACTG CAAAGGCC GGAACGAT TACCATTA CCACGATG ACCACTAA AACGACCG AGATTAAG GGTTTACC GAGTTCAG CCACTGCC ACTATTAA GTGGAAAT TACTTATT AAAGGCAG TTATAAAT GGAAGGGA
GAATCTTT CTACCTGT AATAATGT TGTTCCGT TAGTTCGT TTTATTAA CGTAGATT TTTCTTCC CAACGTCC TGACTGGT ATAATGAG CCAGTTCT TAAAATCG CATAAGGT
TCTGTCGC TACTGATT ACGGTGCT GCTATCGA TGGTTTCA TTGGTGAC GTTTCCGG CCTTGCTA ATGGTAAT GGTGCTAC TGGTGATT TTGCTGGC TCTAATTC CCAAATGG CTCAAGTC GGTGACGG TGATAATT CACCTTTA ATGAATAA TTTCCGTC AATATTTA CCTTCCCT
CAGCAGAC CATTTGCT CCCAATAC TATCACAA CGAGAATG ATACGGAG CATTAAGG AAAACCGC AATACATA GACGTAAT CAACTTAC ACCATAAG GATTTAGA GTTGACTA
GTCGTCTG GTAAACGA GGGTTATG ATAGTGTT GCTCTTAC TATGCCTC GTAATTCC TTTTGGCG TTATGTAT CTGCATTA GTTGAATG TGGTATTC CTAAATCT CAACTGAT
TTATAAAT ACTGCTAA GGCGTCAT AACCTGCG ATAGGTCA GATTTGTA AAATGATA ATGGGGGA GACCGTTT TGAAGAAA ACGTTTTC GGAGAGCG ATAAAACC AAAAATAG
TTAGTACG GTCAAGAA AACCCATA AGGCAATA ATAACGCA AAGGAGCC AAAGGAAG ACCATTGA AACAAGCC GATAGACG AATGAAAA GAATTTTT CCCGAAGC CATTCTAT CGATAACG ATAAAGTA ACAAAGAA CGAGAATA ATAACCCG AATTGAGT TAAGAACA CCCAATAG
AATATTTA TGACGATT CCGCAGTA TTGGACGC TATCCAGT CTAAACAT TTTACTAT TACCCCCT CTGGCAAA ACTTCTTT TGCAAAAG CCTCTCGC TATTTTGG TTTTTATC
AATCATGC CAGTTCTT TTGGGTAT TCCGTTAT TATTGCGT TTCCTCGG TTTCCTTC TGGTAACT TTGTTCGG CTATCTGC TTACTTTT CTTAAAAA GGGCTTCG GTAAGATA GCTATTGC TATTTCAT TGTTTCTT GCTCTTAT TATTGGGC TTAACTCA ATTCTTGT GGGTTATC
ACTACGTT AGGCGAAA CGAAGACT GATATTAT CAGTCCCA TTTCTGGA CTAAAAAC TAAATACC AGTAAGAG CAAAAGAC TTGACAAA TTTCGTAA ACTCCCCC TAAGTTAC
TGATGCAA TCCGCTTT GCTTCTGA CTATAATA GTCAGGGT AAAGACCT GATTTTTG ATTTATGG TCATTCTC GTTTTCTG AACTGTTT AAAGCATT TGAGGGGG ATTCAATG
TTTTTTTT
GCGTTTTT ACTGGAGA ATAGTTTT CCTCGTTA ATTTCCAT GAGAGATT AGGACTGG ACAACCTC AAACGAAG GCCAGACC AAGCGAAA CTTCGAGC TTAATTTT GCGCTATA AACTTCAG AAAGCCCG AAGGAGAA TTAGAAAA
AATAAAAC ATTGACCG TTTAATCC GAGACCTT TCTGCGAG CAATCGCA ACCATTCT AAGTCCTA TTTTAACA TCGACCCA CGTTTTAT CGTTGATT AGAACTAA ATTCCGAA GTTTTGGA GGGCGTTC AGCCCTCC AAGCGATT
CGCAAAAA TGACCTCT TATCAAAA GGAGCAAT TAAAGGTA CTCTCTAA TCCTGACC TGTTGGAG TTTGCTTC CGGTCTGG TTCGCTTT GAAGCTCG AATTAAAA CGCGATAT TTGAAGTC TTTCGGGC TTCCTCTT AATCTTTT
TTATTTTG TAACTGGC AAATTAGG CTCTGGAA AGACGCTC GTTAGCGT TGGTAAGA TTCAGGAT AAAATTGT AGCTGGGT GCAAAATA GCAACTAA TCTTGATT TAAGGCTT CAAAACCT CCCGCAAG TCGGGAGG TTCGCTAA
ATTACCAG TTTGATTT AGATGAGC AAGCGTCT TAACCCTT AGTTGACA ATATACCT TACTTTGA AGGTCTGT GGCATGAA ATCAACGT ATAAATTT TGTACAAC TCGATGTC GTAATATA AGTCGTTA ATTCGAGA TTCGGTAG
TAATGGTC AAACTAAA TCTACTCG TTCGCAGA ATTGGGAA TCAACTGT TATATGGA ATGAAACT TCCAGACA CCGTACTT TAGTTGCA TATTTAAA ACATGTTG AGCTACAG CATTATAT TCAGCAAT TAAGCTCT AAGCCATC
AACGCCTC GCGTTCTT AGAATACC GGATAAGC CTTCTATA TCTGATTT GCTTGCTA TTGGGCGC GGTAATGA TTCCTACG ATGAAAAT AAAAACGG CTTGCTTG TTCTCGAT GAGTGCGG TACTTGGT TTAATACC CGTTCTTG
ATTAAAAC GATTAAGA AACGGAAC GGACATAC TAAATAAC CTACAATT ACGATGAT GATAATCA TCTTAACT ACGGTGGA AAAGTCGA GCGCGGGG TTTACTTT TATATCGA TTTGTCCA ATAACTGG TAAACGCT TTACATAG
CTTACTAT TCCTTTCT GTCGGCTA ATAACTAA CCAAAGAT GTACGAGC ATTTAATC CTACCCTA TAATAAAA AGAACAAG TCCTGAAT AGATAACA ACTATTTG TCCGCGCA AGACGTAA TCGACTTG TACAACAA ATAACAGC
TAATTTTG CTAATTCT TTGCCTTG CCTGTATG ATTTATTG GATGTTAA TGCTACTA CTATTAGT AGAATTGA TGCCACCT TTTCAGCT CGCGCCCC AAATGAAA ATATAGCT AAACAGGT TATTGACC ATTTGCGA AATGTATC
GAATGATA AGGAAAGA CAGCCGAT TATTGATT GGTTTCTA CATGCTCG TAAATTAG GATGGGAT ATTATTTT TCTTGTTC AGGACTTA TCTATTGT TGATAAAC AGGCGCGT TCTGCATT AGCTGAAC ATGTTGTT TATTGTCG
AGCAGACC TGTCTTAA TGAAATGG AAAACAGC CATGAAAT ATAAGAGA ATAATGAC CGAGCTTT TACGGAGA CGGATTTA ATGTACAA CCGCAACA ATTTATAC CGCTAAGA GTTAATTC GGGATGAC AACTCGCA ACCGAAAT
GAGTCCGT AACGTAAA TTTTATAT ACTCCCAA GATTTTTA AAAATAGG AACGCAAC TTTATTTC CGAAGAGG GCGTTTTC ATAATGTC CCAGTATT ACAAAAAC CATGTTGG CTAAATCG AAATACGA GACTCCGA AATAACGA
CTCAGGCA TTGCATTT AAAATATA TGAGGGTT CTAAAAAT TTTTATCC TTGCGTTG AAATAAAG GCTTCTCC CGCAAAAG TATTACAG GGTCATAA TGTTTTTG GTACAACC GATTTAGC TTTATGCT CTGAGGCT TTATTGCT
ACTGGACT ATCGGAAA CATCTAGA GAGTTTTT ATCGATGG GAGAGGCC GTAATTAA ATAGTCGA TCTTGCCA ACTTATAG TATAACTA CCACTAAA CTGACAGA GGCCGGAA AGAGTGGG AAAACTTA GAAATGGA TGTGTAAT
ATGACCAT TCTTAAAC ATATTGCG TATACTAT GATTTGTC CGAAAAAG ATCATTAA TACTAAGG CCACAAAT AAGAATAA ATTGCGGA ATAAATAG TGTGCCAG CCATAAAG TTTGGTAA TTTAAATC CAGTCTTC TACTTTAA
TGACCTGA TAGCCTTT GTAGATCT CTCAAAAA TAGCTACC CTCTCCGG CATTAATT TATCAGCT AGAACGGT TGAATATC ATATTGAT GGTGATTT GACTGTCT CCGGCCTT TCTCACCC TTTTGAAT CTTTACCT ACACATTA
TACTGGTA AGAATTTG TATAACGC ATATGATA CTAAACAG GCTTTTTC TAGTAATT ATGATTCC GGTGTTTA TTCTTATT TAACGCCT TATTTATC ACACGGTC GGTATTTC AAACCATT AAATTTAG GTCAGAAG ATGAAATT
TTGATTTT ATATAAAC TTTTTCAA AAGAGCGC AAGAAACA GAACGCTA ACCTAAAC GTAGTCGT AAATGTAT ATCAATAT ATTGGGTT GGATTCGG CCTCCAAT TTTTCCAT CAGAGAGT CTGGATAC TAAAACTA TTTAAGTG
TTGCAAAT GTTAAATT TATAAACG AATATGTT AGAAGGAC AAAAACCC CGAAAAGA CTAATAGT TGGCCCCA TGTATACT AACTGTAC GATCAAAA TGCTAATG GCAAGTAG CTAAGAGA ACAAACGA GGTCTGAG AGTCCGTT
AACGTTTA CAATTTAA ATATTTGC TTATACAA TCTTCCTG TTTTTGGG GCTTTTCT GATTATCA ACCGGGGT ACATATGA TTGACATG CTAGTTTT ACGATTAC CGTTCATC GATTCTCT TGTTTGCT CCAGACTC TCAGGCAA
ATAACTGA GAAGAGTC GCAGAATT AGATTCGA TAGCGATA CAAAAGTT CCTAAGAT TCCCTTTT AATTAATT ATCGCTGC TAAATGTC TTCGTTCC AATAAGTG AGTGTATA TAACTAAA TACATGAC AAAGGTAA TTTTTTCC
CGACGGGT TGTTACTC GCTCACAT TTAATGTT GATGAAAG CTGGCTAC AGGAAGGC CAGACGCG AATTATTT TTGATGGC GTTCCTAT TGGTTAAA AAATGAGC TGATTTAA CAAAAATT TAATGCGA ATTTTAAC AAAATATT
TATTGACT CTTCTCAG CGTCTTAA TCTAAGCT ATCGCTAT GTTTTCAA GGATTCTA AGGGAAAA TTAATTAA TAGCGACG ATTTACAG AAGCAAGG TTATTCAC TCACATAT ATTGATTT ATGTACTG TTTCCATT AAAAAAGG
ATTAAGTT TACTTTAA CAATTTAC ATTAATTA AAACAAAA GAACTACA AACAAAGT AGTAGAAG AAAACGAG TCCATTAA CTTTACTT ATTAAGCG GAGACGCG CTAAAACA TTGAACCA TAAGTTTC GTTAGTCC GCTTAGGC
TTTCGACC GACCTCAC GCTAGAAG GACTCCGG CTATGACA GCAGCAGG GGAGTTTG ACCGTCTA CGTGCCAA TGCTACGC GGGTAGAT GTGGTTGC ACTGGATA GGGTAATG CCAGTTAG GCGGCAAA CAAGGGTG CCTCTTAG
AAAGCTGG CTGGAGTG CGATCTTC CTGAGGCC GATACTGT CGTCGTCC CCTCAAAC TGGCAGAT GCACGGTT ACGATGCG CCCATCTA CACCAACG TGACCTAT CCCATTAC GGTCAATC CGCCGTTT GTTCCCAC GGAGAATC
AATAACAA AGAGGGCT ACATTTTC CATGACAA TGACATAT AAGTAGAC TGCAATTT GGACTTTT AGATGCGT TAAAGAAA TAAAGACA AAATGCAC GTTTATTA AAACTATA CCATCCAA GATTGGGA AGGTAATA AGTCTTCA
AACTTAAT CGCCTTGC AGCACATC CCCCTTTC GCCAGCTG GCGTAATA GCGAAGAG GCCCGCAC CGATCGCC CTTCCCAA CAGTTGCG CAGCCTGA ATGGCGAA TGGCGCTT TGCCTGGT TTCCGGCA CCAGAAGC GGTGCCGG
TTATTGTT TCTCCCGA TGTAAAAG GTACTGTT ACTGTATA TTCATCTG ACGTTAAA CCTGAAAA TCTACGCA ATTTCTTT ATTTCTGT TTTACGTG CAAATAAT TTTGATAT GGTAGGTT CTAACCCT TCCATTAT TCAGAAGT
TATTAGGT TTGTTAGT CCTAATAT AACTACTT AACGGTAG TAGACTAT TAGTCCTT ATACTACT ATTAAGGC GAGGAAGA CCACCAAA GAAACAAG GCGTTTTA CTATTACA ATGAGTTT GAAAATTT TAATTATT GCAAGCCC
TAAAGTGT GTCCTTTG TCGATACT GGTACTAA TGCTTAAG CTCGAGCC ATGGGCCC CTAGGAGA TCTCAGCT GGACGTCC GTACGTTC GAACCGTG ACCGGCAG CAAAATGT TGCAGCAC TGACCCTT TTGGGACC GCAATGGG
ATTTCACA CAGGAAAC AGCTATGA CCATGATT ACGAATTC GAGCTCGG TACCCGGG GATCCTCT AGAGTCGA CCTGCAGG CATGCAAG CTTGGCAC TGGCCGTC GTTTTACA ACGTCGTG ACTGGGAA AACCCTGG CGTTACCC
CGAAATGT GAAATACG AAGGCCGA GCATACAA CACACCTT AACACTCG CCTATTGT
GTTTCCTA AATTATGC TCAACAGC TTAACAAA CATTTCAG ATTATGAA GATTTAGG AGTTTACA TAATAGAT
GACAACGT CCGCCACA ATTATGAC TGGCGGAG TGGAGACA AAATAGAA GACGACCA CCAAGCAA GCCATAAA
CCTGCTGG GGCAAACC AGCGTGGA CCGCTTGC TGCAACTC TCTCAGGG CCAGGCGG TGAAGGGC AATCAGCT GTTGCCCG TCTCACTG
GCTTTACA CTTTATGC TTCCGGCT CGTATGTT GTGTGGAA TTGTGAGC GGATAACA
CAAAGGAT TTAATACG AGTTGTCG AATTGTTT GTAAAGTC TAATACTT CTAAATCC TCAAATGT ATTATCTA
CTGTTGCA GGCGGTGT TAATACTG ACCGCCTC ACCTCTGT TTTATCTT CTGCTGGT GGTTCGTT CGGTATTT
AAGCGGGA AACTGCAA CCTCAGGT GCAAGAAA TTATCACC TGAGAACA AGGTTTGA CCTTGTTG TGAGTTGG GATAGAGC CCGATAAG AAAACTAA ATATTCCC TAAAACGG CTAAAGCC TTGGTGGT AGTTTGTC CTAAAAGC
CTGGGGTT TTTTGAAC TAAACCCA CTACCAAG TGCATCAC CCGGTAGC GGGACTAT CTGCCAAA
TTCGCCCT TTGACGTT GGAGTCCA CGTTCTTT AATAGTGG ACTCTTGT TCCAAACT GGAACAAC ACTCAACC CTATCTCG GGCTATTC TTTTGATT TATAAGGG ATTTTGCC GATTTCGG AACCACCA TCAAACAG GATTTTCG
GACCCCAA AAAACTTG ATTTGGGT GATGGTTC ACGTAGTG GGCCATCG CCCTGATA GACGGTTT
TTAATGGC GATGTTTT AGGGCTAT CAGTTCGC GCATTAAA GACTAATA GCCATTCA AAAATATT GTCTGTGC CACGTATT
GGTGCAAG CGGCCGAA AGGGGCAG TTCGAGAT TTAGCCCC CGAGGGAA ATCCCAAG GCTAAATC ACGAAATG CCGTGGAG
GAATGCGA AAGTCCAG TCTTCCCA AGATAGAG ACAACCGG TCTTACAG GGAAAATA ATGACCAG CACACTGA CCACTTAG
CCACGTTC GCCGGCTT TCCCCGTC AAGCTCTA AATCGGGG GCTCCCTT TAGGGTTC CGATTTAG TGCTTTAC GGCACCTC
CTTACGCT TTCAGGTC AGAAGGGT TCTATCTC TGTTGGCC AGAATGTC CCTTTTAT TACTGGTC GTGTGACT GGTGAATC
AATTACCG CTACAAAA TCCCGATA GTCAAGCG CGTAATTT CTGATTAT CGGTAAGT TTTTATAA CAGACACG GTGCATAA
ACGGTTAC ATTTATTA GGTAAAGT CTGCTAAC TCGCAGTT TTACATCC ATAAAGGT ACTCGCAA AAAGGACA ACGTTACC
CCACCAAT GCGCGTCG CACTGGCG ATGTGAAC GGTCGCGG GATCGCGG GCGAGGAA AGCGAAAG AAGGGAAG GAAAGAGC
GGTGGTTA CGCGCAGC GTGACCGC TACACTTG CCAGCGCC CTAGCGCC CGCTCCTT TCGCTTTC TTCCCTTC CTTTCTCG
TGCTCCTT TCGTGCAA TATGCACG AGCAGTTT CGTTGGTA TCATGCGC GGGACATC GCCGCGTA ATTCGCGC CGCCCACA
GACCGCCA TTATAACA AGACCTAT AATGGTCG TTCCGGCT ATCAAACT CAAGAAGA TGAGTCCG TTCACTAC AATAATGA
ACGAGGAA AGCACGTT ATACGTGC TCGTCAAA GCAACCAT AGTACGCG CCCTGTAG CGGCGCAT TAAGCGCG GCGGGTGT
CTGGCGGT AATATTGT TCTGGATA TTACCAGC AAGGCCGA TAGTTTGA GTTCTTCT ACTCAGGC AAGTGATG TTATTACT
TTAGTTTC TTCATAAC GATGTTGC CAATTAAA CGCACTAC CTGTCTGA GAAAATGA GCCACCGG AGTGACTA ATATTTTT
GTGAAGAG TCCTAAGA CCGCATGG CAAGGACA GATTTTAG GGAAATTA GCCGGAGG ACAAATCG AGGGCGAG ACTAAGAT
AATCAAAG AAGTATTG CTACAACG GTTAATTT GCGTGATG GACAGACT CTTTTACT CGGTGGCC TCACTGAT TATAAAAA -- CACTTCTC AGGATTCT GGCGTACC GTTCCTGT CTAAAATC CCTTTAAT CGGCCTCC TGTTTAGC TCCCGCTC TGATTCTA
AACTGCCG AGATTAGA TAATCAAC AATCACGA
TACACTCA ATCGAGTG AGTAATCC GTGGGGTC
TTGACGGC TCTAATCT ATTAGTTG TTAGTGCT CCTAAAGATATT
GGAGTTAA GGAAAGTT GACAACTA AACGGTTG
AAAAAGTA AACGACGA CCGAGAGT CGCACCGT
CGCAATTA ATGTGAGT TAGCTCAC TCATTAGG CACCCCAG
MR
TGATGCTTTAGA TTTTTCAT TTGCTGCT GGCTCTCA GCGTGGCA
TGCTGTCC AAAGGGCT GACCTTTC GCCCGTCA
K
L
M
TGCCAATG TAAATAAT CCATTTCA GACGATTG AGCGTCAA AATGTAGG TATTTCCA TGAGCGTT TTTCCTGT TGCAATGG
CACTTTTC TTTTTGGT GGGACCGC GGGTTATG
GTGAAAAG AAAAACCA CCCTGGCG CCCAATAC GCAAACCG
GCGCGCAA CCGGCTAA GTAATTAC GTCGACCG
CCTCTCCC CGCGCGTT GGCCGATT CATTAATG CAGCTGGC -- ACGACAGG TTTCCCGA CTGGAAAG CGGGCAGT GAGCGCAA
J
ATAATCCA AACAATCA GGATTATA TTGATGAA TTGCCATC ATCTGATA ATCAGGAA TATGATGA TAATTCCG CTCCTTCT GGTGGTTT CTTTGTTC CGCAAAAT GATAATGT TACTCAAA CTTTTAAA ATTAATAA CGTTCGGG
GGACGACC CCGTTTGG TCGCACCT GGCGAACG ACGTTGAG AGAGTCCC GGTCCGCC ACTTCCCG TTAGTCGA CAACGGGC AGAGTGAC
ML
I
TAATTCAA ATGAAATT GTTAAATG TAATTAAT TTTGTTTT CTTGATGT TTGTTTCA TCATCTTC TTTTGCTC AGGTAATT GAAATGAA TAATTCGC CTCTGCGC GATTTTGT AACTTGGT ATTCAAAG CAATCAGG CGAATCCG
TTGAATTA GCGGAACG TCGTGTAG GGGGAAAG CGGTCGAC CGCATTAT CGCTTCTC CGGGCGTG GCTAGCGG GAAGGGTT GTCAACGC GTCGGACT TACCGCTT ACCGCGAA ACGGACCA AAGGCCGT GGTCTTCG CCACGGCC
N
H
AACTAAAA TATATTTG AAAAAGTT TTCTCGCG TTCTTTGT CTTGCGAT TGGATTTG CATCAGCA TTTACATA TAGTTATA TAACCCAA CCTAAGCC GGAGGTTA AAAAGGTA GTCTCTCA GACCTATG ATTTTGAT AAATTCAC
GCTGCCCA ACAATGAG CGAGTGTA AATTACAA CTACTTTC GACCGATG TCCTTCCG GTCTGCGC TTAATAAA AACTACCG CAAGGATA ACCAATTT TTTACTCG ACTAAATT GTTTTTAA ATTACGCT TAAAATTG TTTTATAA
M
G
TCGTCTGG ACAGAATT ACTTTACC TTTTGTCG GTACTTTA TATTCTCT TATTACTG GCTCGAAA ATGCCTCT GCCTAAAT TACATGTT GGCGTTGT TAAATATG GCGATTCT CAATTAAG CCCTACTG TTGAGCGT TGGCTTTA
L
TTGCGGAG CGCAAGAA TCTTATGG CCTATTCG GAAGATAT AGACTAAA CGAACGAT AACCCGCG CCATTACT AAGGATGC TACTTTTA TTTTTGCC GAACGAAC AAGAGCTA CTCACGCC ATGAACCA AATTATGG GCAAGAAC
K
TCTCTGAT ATTAGCGC TCAATTAC CCTCTGAC TTTGTTCA GGGTGTTC AGTTAATT CTCCCGTC TAATGCGC TTCCCTGT TTTTATGT TATTCTCT CTGTAAAG GCTGCTAT TTTCATTT TTGACGTT AAACAAAA AATCGTTT CTTATTTG GATTGGGA TAAATAAT ATGGCTGT
J
AGAGACTA TAATCGCG AGTTAATG GGAGACTG AAACAAGT CCCACAAG TCAATTAA GAGGGCAG ATTACGCG AAGGGACA AAAATACA ATAAGAGA GACATTTC CGACGATA AAAGTAAA AACTGCAA TTTGTTTT TTAGCAAA GAATAAAC CTAACCCT ATTTATTA TACCGACA
I
E
GGGAGTTA GCCAACTT ACAGCGGG AAAACAGA AACCGCGA CCATTTGG TATACTTA AAAGATAA CTAACACT GTTTTATT TGAATAAG GCACCACA GAAACGCA AAGAAAAT ATACAACG GTGGAAAT ACATACAT AAAAGATG CAAACGAT TGTATGAC GCATTATT CCTCAGAA
H
D
CTTAGAAA GATGGACA TTATTACA ACAAGGCA ATCAAGCA AAATAATT GCATCTAA AAAGAAGG GTTGCAGG ACTGACCA TATTACTC GGTCAAGA ATTTTAGC GTATTCCA
CCCTCAAT CGGTTGAA TGTCGCCC TTTTGTCT TTGGCGCT GGTAAACC ATATGAAT TTTCTATT GATTGTGA CAAAATAA ACTTATTC CGTGGTGT CTTTGCGT TTCTTTTA TATGTTGC CACCTTTA TGTATGTA TTTTCTAC GTTTGCTA ACATACTG CGTAATAA GGAGTCTT
TTTTTTTT
F
G
C
TTTTTTTT
E
TTTTTTTT
JP- 1
Staple strands indicated by bold lines are hairpin-incorporated strands.
GGGGCATT AACTGTTT ATACGGGC ACTGTTAC TCAAGGCA CTGACCCC GTTAAAAC TTATTACC AGTACACT CCTGTATC ATCAAAAG CCATGTAT GACGCTTA CTGGAACG GTAAATTC AGAGACTG CGCTTTCC ATTCTGGC
D
CGTTCGGA GTCGCTGG CTTATATA GCCAATAC GCACCCGC TACCAACA
C
AATAATAA GCGTTAAG GAAATCAA CAAGGAAA GATAAGAG TGAGGCGA CTTTGACA ACTTTCAA CAAATCGT TTTAGGGT
B
TTATTATT CGCAATTC CTTTAGTT GTTCCTTT CTATTCTC ACTCCGCT GAAACTGT TGAAAGTT GTTTAGCA AAATCCCA
A
ACTGGTCT ATAACTAA CTCCCAAA CTATAAAC
TTAGATAACCTT CCTCAATT CCTTTCAA CTGTTGAT TTGCCAAC -- TGACCAGA TATTGATT GAGGGTTT GATATTTG AGGTTCAGCAAGG
Fig. S1. The design of the jigsaw-shaped DNA origami (JP-1) which is used in this study.
N
F
Polymer : M13 = 0.5 : 1
Polymer : M13 = 1 : 1
Polymer : M13 = 1.25 : 1
PVS added
Lock M13mp18 and
release using PVS
1500 × 1125 nm
N/P = 0.2
1400 × 1050 nm N/P = 0.25
2480 × 1860 nm
1400 × 1050 nm
1800 × 1350 nm
No PVS
i) Mix M13mp18 + Buffer + 8K90D
N/P = 0.1
ii) Incubate at room temp for 30 min
iii) Add Staple strands
iv) Add PVS / For control don’t add PVS
v) Anneal 85-15°C (-1°C/min)
vi) Purify using S-500
vii) AFM imaging
1500 × 1125 nm
Polymer : M13 = 2 : 1
Polymer : M13 = 3 : 1
Polymer : M13 = 4 : 1
PVS added
Polymer : M13 = 1.5 : 1
1500 × 1125 nm N/P = 0.4
2400 × 1800 nm
2480 × 1860 nm
2400 × 1800 nm
N/P = 0.6
2000 × 1500 nm N/P = 0.8
2400 × 1800 nm
1000 × 750 nm
2400 × 1800 nm
No PVS
N/P = 0.3
Fig. S2. AFM images of the DNA origami formation when M13 DNA was locked using 8K90D
and either released using PVS (sample) or no PVS was used (control). The experiments were
performed with different N/P ratio. Image sizes are given at the bottom right corner of each image.
[M13mp18] = 10 nM; [Staples] = 40 nM; [Tris-HCl] = 20 mM, pH 7.6; [MgCl2] = 10 mM; [EDTA]
= 1 mM; Polymer : PVS = 1 : 5; Annealing: 85-15ºC (-1ºC/min).
Polymer : Staples = 0.25 : 1
Polymer : Staples = 0.5 : 1
PVS added
Lock staple strands and
release using PVS
N/P = 0.2
1600 × 1200 nm
N/P = 0.4
1200 × 900 nm
No PVS
i) Mix Staples + Buffer + 8K90D
ii) Incubate at room temp for 30 min
iii) Add M13mp18
iv) Add PVS / For control don’t add PVS
v) Anneal 85-15°C (-1°C/min)
vi) Purify using S-500
vii) AFM imaging
2000 × 1500 nm
Polymer : Staples = 1.5 : 1
Polymer : Staples = 2 : 1
PVS added
Polymer : Staples = 1 : 1
2480 × 1860 nm
2380 × 1785 nm
N/P = 1.2
2480 × 1860 nm
N/P = 1.6
2480 × 1860 nm
No PVS
N/P = 0.8
1400 × 1050 nm
2480 × 1860 nm
2480 × 1860 nm
Fig. S3. AFM images of the DNA origami formation when staple strands were locked using 8K90D
and either released using PVS (sample) or no PVS was used (control). The experiments were
performed with different N/P ratio. Image sizes are given at the bottom right corner of each image.
[M13mp18] = 10 nM; [Staples] = 40 nM; [Tris-HCl] = 20 mM, pH 7.6; [MgCl2] = 10 mM; [EDTA]
= 1 mM; Polymer : PVS = 1 : 5; Annealing: 85-15ºC (-1ºC/min).
Lock both M13mp18 and staple strands at the same time and release using PVS
Polymer : (M13 + Staples) = 0.5 : 1
Polymer : (M13 + Staples) = 1 : 1
PVS added
Polymer : (M13 + Staples) = 0.25 : 1
N/P = 0.25
1500 × 1125 nm
N/P = 0.5
2400 × 1800 nm N/P = 1
Polymer : (M13 + Staples) = 0.5 : 1
1600 × 1200 nm
Polymer : (M13 + Staples) = 1 : 1
No PVS
Polymer : (M13 + Staples) = 0.25 : 1
1200 × 900 nm
2000 × 1500 nm
1200 × 900 nm
i) Mix M13mp18 + Staples + Buffer + 8K90D
ii) Incubate at room temp for 30 min
iii) Add PVS / For control don’t add PVS
iv) Anneal 85-15°C (-1°C/min)
v) Purify using S-500
vi) AFM imaging
Fig. S4. AFM images of the DNA origami formation when both M13 and staples were locked using
8K90D and either released using PVS (sample) or no PVS was used (control). The experiments
were performed with different N/P ratio. Image sizes are given at the bottom right corner of each
image. [M13mp18] = 10 nM; [Staples] = 40 nM; [Tris-HCl] = 20 mM, pH 7.6; [MgCl2] = 10 mM;
[EDTA] = 1 mM; Polymer : PVS = 1 : 5; Annealing: 85-15ºC (-1ºC/min).
Lock M13
Polymer : M13 = 5 : 1
Lock staples
Polymer : Staples = 1.25 : 1
Lock both M13 and staples
Polymer : (M13 + Staples) = 1 : 1
Fig. S5. Representative zoom-in AFM images of the DNA origami formation when ssDNAs were
locked using 15K92D and released using PVS. All experimental conditions are same as given in
main text Fig. 3. Image size: 400 nm × 300 nm.
Table S1. The yield of the jigsaw-shaped origami structure at different conditions
Polymer
8K90D
8K90D
8K90D
15K92D
15K92D
15K92D
Lock
M13
Staples
Both
M13
Staples
Both
Polymer:DNA
2:1
0.5:1
0.5:1
5:1
1.25:1
1:1
N/P Ratio
0.4
0.4
0.5
1
1
1
Yield
90%
86%
72%
95%
90%
86%
Number of Tiles Calculated
202
165
79
144
208
234
All experimental conditions are same as given in main text Fig. 2 and 3.
Simultaneous addition of polymer and PVS
8K90D : (M13 + Staples) = 1 : 1
N/P = 1
2480 × 1860 nm
15K92D : (M13 + Staples) = 1 : 1
N/P = 1
1200 × 900 nm
i) Mix M13mp18 + Staples + Buffer + 8K90D/15K92D + PVS
ii) Incubate at room temp for 30 min
iii) Anneal 85-15°C (-1°C/min)
iv) Purify using S-500
v) AFM imaging
Fig. S6. AFM images of the DNA origami formation when CCC (left: 8K90D and right: 15K92D)
and PVS were simultaneously added. Image sizes are given at the bottom right corner of each image.
[M13mp18] = 10 nM; [Staples] = 40 nM; [Tris-HCl] = 20 mM, pH 7.6; [MgCl2] = 10 mM; [EDTA]
= 1 mM; N/P = 1; Polymer : PVS = 1 : 5; Annealing: 85-15ºC (-1ºC/min).
Lock both M13 and staples
Polymer : M13 = 5 : 1
Polymer : Staples = 1.25 : 1
Polymer : (M13 + Staples) = 1 : 1
N/P = 1
2480 × 1860 nm
N/P = 1
2480 × 1860 nm
2480 × 1860 nm
2480 × 1860 nm
2480 × 1860 nm
40 mM Phosphate buffer
Polymer : Phosphate Buf = 1 : 110
1400 × 1050 nm
No PVS
N/P = 1
20 mM Phosphate buffer
Polymer : Phosphate Buf = 1 : 55
Lock staples
No PVS
Lock M13
i) Mix M13mp18 + Phosphate buffer + 8K90D
ii) Incubate at room temp for 30 min
iii) Add staple strands
iv) Anneal 85-15°C (-1°C/min)
v) Purify using S-500
vi) AFM imaging
Fig. S7. AFM images of the DNA origami formation when either M13/staples alone or both
together locked using 8K90D and the experiments were performed in phosphate buffer. No PVS
was added in these experiments. Image sizes are given at the bottom right corner of each image.
[M13mp18] = 10 nM; [Staples] = 40 nM; [MgCl2] = 10 mM; [EDTA] = 1 mM; Buffer pH 7.6; N/P
= 1; Annealing: 85-15ºC (-1ºC/min).
Lock staples
Lock both M13 and staples
Polymer : M13 = 5 : 1
Polymer : Staples = 1.25 : 1
Polymer : (M13 + Staples) = 1 : 1
No PVS
2 mM NTPs
Polymer : NTPs = 1 : 16.5
Lock M13
2480 × 1860 nm
N/P = 1
2480 × 1860 nm
N/P = 1
2480 × 1860 nm
No PVS
5.64 mM NTPs
Polymer : NTPs = 1 : 46.6
N/P = 1
2480 × 1860 nm
2480 × 1860 nm
2480 × 1860 nm
i) Mix M13mp18 + Buffer + NTPs + 8K90D
ii) Incubate at room temp for 30 min
iii) Add staple strands
iv) Anneal 85-15°C (-1°C/min)
v) Purify using S-500
vi) AFM imaging
Fig. S8. AFM images of the DNA origami formation when either M13/staples alone or both
together locked using 8K90D and the experiments were performed in the presence of NTPs. No
PVS was added in these experiments. Image sizes are given at the bottom right corner of each
image. [M13mp18] = 10 nM; [Staples] = 40 nM; [Tris-HCl] = 20 mM, pH 7.6; [MgCl2] = 10 mM;
[EDTA] = 1 mM; N/P = 1; Annealing: 85-15ºC (-1ºC/min).