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

aDepartment of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan. Email: hs@kuchem.kyoto-u.ac.jp

bCurrent addresses: Faculty of Medicine and Life Science Center of TARA, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-shi, Ibaraki-ken 305-8577, Japan

cInstitute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan. Email: endo@kuchem.kyoto-u.ac.jp

dCREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan eDepartment 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

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2014

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.

Fig. S1. The design of the jigsaw-shaped DNA origami (JP-1) which is used in this study.

TTTCGCTG CTGAGGGT GACGATCC CGCAAAAG CGGCCTTT AACTCCCT

TTTAATGA GGATTTAT TTGTTTGT GAATATCA AGGCCAAT CGTCTGAC CTGCCTCA ACCTCCTG TCAATGCT GGCGGCGG CTCTGGTG GTGGTTCT GGTGGCGG CTCTGAGG GTGGTGGC TCTGAGGG TGGCGGTT CTGAGGGT

GAATGATA AGGAAAGA CAGCCGAT TATTGATT GGTTTCTA CATGCTCG TAAATTAG GATGGGAT ATTATTTT TCTTGTTC AGGACTTA TCTATTGT TGATAAAC AGGCGCGT TCTGCATT AGCTGAAC ATGTTGTT TATTGTCG

TACTGGTA AGAATTTG TATAACGC ATATGATA CTAAACAG GCTTTTTC TAGTAATT ATGATTCC GGTGTTTA TTCTTATT TAACGCCT TATTTATC ACACGGTC GGTATTTC AAACCATT AAATTTAG GTCAGAAG ATGAAATT

TATTGACT CTTCTCAG CGTCTTAA TCTAAGCT ATCGCTAT GTTTTCAA GGATTCTA AGGGAAAA TTAATTAA TAGCGACG ATTTACAG AAGCAAGG TTATTCAC TCACATAT ATTGATTT ATGTACTG TTTCCATT AAAAAAGG

TTATTGTT TCTCCCGA TGTAAAAG GTACTGTT ACTGTATA TTCATCTG ACGTTAAA CCTGAAAA TCTACGCA ATTTCTTT ATTTCTGT TTTACGTG CAAATAAT TTTGATAT GGTAGGTT CTAACCCT TCCATTAT TCAGAAGT

CAAAGGAT TTAATACG AGTTGTCG AATTGTTT GTAAAGTC TAATACTT CTAAATCC TCAAATGT ATTATCTA

TTAATGGC GATGTTTT AGGGCTAT CAGTTCGC GCATTAAA GACTAATA GCCATTCA AAAATATT GTCTGTGC CACGTATT

AATCAAAG AAGTATTG CTACAACG GTTAATTT GCGTGATG GACAGACT CTTTTACT CGGTGGCC TCACTGAT TATAAAAA --CACTTCTC AGGATTCT GGCGTACC GTTCCTGTCTAAAATC CCTTTAAT CGGCCTCC TGTTTAGC TCCCGCTC TGATTCTA

GCTTTACA CTTTATGC TTCCGGCT CGTATGTT GTGTGGAA TTGTGAGC GGATAACA

ATTTCACA CAGGAAAC AGCTATGA CCATGATT ACGAATTC GAGCTCGG TACCCGGG GATCCTCT AGAGTCGA CCTGCAGG CATGCAAG CTTGGCAC TGGCCGTC GTTTTACAACGTCGTG ACTGGGAA AACCCTGG CGTTACCC

CGACGGGT TGTTACTC GCTCACAT TTAATGTT GATGAAAG CTGGCTAC AGGAAGGC CAGACGCG AATTATTT TTGATGGC GTTCCTAT TGGTTAAA AAATGAGC TGATTTAA CAAAAATT TAATGCGA ATTTTAAC AAAATATT

TGACCTGA TAGCCTTT GTAGATCT CTCAAAAA TAGCTACC CTCTCCGG CATTAATT TATCAGCT AGAACGGT TGAATATC ATATTGAT GGTGATTT GACTGTCT CCGGCCTT TCTCACCC TTTTGAAT CTTTACCT ACACATTA

TGATGCAA TCCGCTTT GCTTCTGA CTATAATA GTCAGGGT AAAGACCT GATTTTTG ATTTATGG TCATTCTC GTTTTCTG AACTGTTT AAAGCATT TGAGGGGG ATTCAATG

GTCGTCTG GTAAACGA GGGTTATG ATAGTGTT GCTCTTAC TATGCCTC GTAATTCC TTTTGGCG TTATGTAT CTGCATTA GTTGAATG TGGTATTC CTAAATCT CAACTGAT

AATTCACA ATGATTAA AGTTGAAA TTAAACCA TCTCAAGC CCAATTTA CTACTCGT TCTGGTGT TTCTCGTC AGGGCAAG CCTTATTC ACTGAATG AGCAGCTT TGTTACGTTGATTTGG GTAATGAA TATCCGGT TCTTGTCA

GATTTCGA CACAATTT ATCAGGCG ATGATACA AATCTCCG TTGTACTT TGTTTCGC GCTTGGTA TAATCGCT GGGGGTCA AAGATGAG TGTTTTAG TGTATTCT TTTGCCTCTTTCGTTT TAGGTTGG TGCCTTCGTAGTGGCA

AGTCCTCA AAGCCTCT GTAGCCGT TGCTACCC TCGTTCCG ATGCTGTC

ACAGTAAC AGCCGCGT TGATAGCC ATAGTTCG ACAAATTC TTTAAGTG GAGCTTTC GTTCGACT ATTTGGCT ATGTTAAT TTCCGAGG AAAACCTC GGAAAAAA AACCTCTA AAAGTTGC ACTTTTTT

1 2 3 4 5 6 7 8

TCAGGAGT TTCGGAGACATCGGCA ACGATGGGAGCAAGGC TACGACAG

CTAAAGCT GTGTTAAATAGTCCGC TACTATGTTTAGAGGC AACATGAAACAAAGCG CGAACCATATTAGCGA CCCCCAGTTTCTACTC ACAAAATCACATAAGA AAACGGAGAAAGCAAA ATCCAACCACGGAAGC ATCACCGT

TTAAGTGT TACTAATTTCAACTTT AATTTGGTAGAGTTCG GGTTAAATGATGAGCA AGACCACAAAGAGCAG TCCCGTTCGGAATAAG TGACTTACTCGTCGAA ACAATGCAACTAAACC CATTACTTATAGGCCA AGAACAGT

CAGCAGAC CATTTGCTCCCAATAC TATCACAACGAGAATG ATACGGAGCATTAAGG AAAACCGCAATACATA GACGTAATCAACTTAC ACCATAAGGATTTAGA GTTGACTA

ACTACGTT AGGCGAAACGAAGACT GATATTATCAGTCCCA TTTCTGGACTAAAAAC TAAATACCAGTAAGAG CAAAAGACTTGACAAA TTTCGTAAACTCCCCC TAAGTTAC

ATTAAAAC GATTAAGA AACGGAAC GGACATAC TAAATAAC CTACAATT ACGATGAT GATAATCA TCTTAACT ACGGTGGA AAAGTCGA GCGCGGGG TTTACTTT TATATCGA TTTGTCCA ATAACTGG TAAACGCT TTACATAG

TAATTTTG CTAATTCT TTGCCTTG CCTGTATG ATTTATTG GATGTTAA TGCTACTA CTATTAGT AGAATTGA TGCCACCT TTTCAGCT CGCGCCCC AAATGAAA ATATAGCT AAACAGGT TATTGACC ATTTGCGA AATGTATC

ACTGGACT ATCGGAAA CATCTAGA GAGTTTTT ATCGATGG GAGAGGCC GTAATTAA ATAGTCGA TCTTGCCA ACTTATAG TATAACTA CCACTAAA CTGACAGA GGCCGGAA AGAGTGGG AAAACTTA GAAATGGA TGTGTAAT

GCTGCCCA ACAATGAG CGAGTGTA AATTACAA CTACTTTC GACCGATG TCCTTCCG GTCTGCGC TTAATAAA AACTACCG CAAGGATA ACCAATTT TTTACTCG ACTAAATT GTTTTTAA ATTACGCT TAAAATTG TTTTATAA

TAAAGTGT GTCCTTTGTCGATACT GGTACTAATGCTTAAG CTCGAGCCATGGGCCC CTAGGAGATCTCAGCT GGACGTCCGTACGTTC GAACCGTGACCGGCAG CAAAATGTTGCAGCAC TGACCCTTTTGGGACC GCAATGGG

CGAAATGT GAAATACG AAGGCCGA GCATACAA CACACCTT AACACTCG CCTATTGT

TTAGTTTC TTCATAACGATGTTGC CAATTAAACGCACTAC CTGTCTGAGAAAATGA GCCACCGGAGTGACTA ATATTTTTGTGAAGAG TCCTAAGACCGCATGG CAAGGACAGATTTTAG GGAAATTAGCCGGAGG ACAAATCGAGGGCGAG ACTAAGAT

AAATTACT CCTAAATA AACAAACA CTTATAGT TCCGGTTA GCAGACTG GACGGAGT TGGAGGAC AGTTACGA CCGCCGCC GAGACCAC CACCAAGA CCACCGCC GAGACTCC CACCACCG AGACTCCC ACCGCCAA GACTCCCA

CTTACTAT TCCTTTCT GTCGGCTA ATAACTAA CCAAAGAT GTACGAGC ATTTAATC CTACCCTA TAATAAAA AGAACAAG TCCTGAAT AGATAACA ACTATTTG TCCGCGCA AGACGTAA TCGACTTG TACAACAA ATAACAGC

ATGACCAT TCTTAAAC ATATTGCG TATACTAT GATTTGTC CGAAAAAG ATCATTAA TACTAAGG CCACAAAT AAGAATAA ATTGCGGA ATAAATAG TGTGCCAG CCATAAAG TTTGGTAA TTTAAATC CAGTCTTC TACTTTAA

ATAACTGA GAAGAGTC GCAGAATT AGATTCGA TAGCGATA CAAAAGTT CCTAAGAT TCCCTTTT AATTAATT ATCGCTGC TAAATGTC TTCGTTCC AATAAGTG AGTGTATA TAACTAAA TACATGAC AAAGGTAA TTTTTTCC

AATAACAA AGAGGGCT ACATTTTC CATGACAA TGACATAT AAGTAGAC TGCAATTT GGACTTTT AGATGCGT TAAAGAAA TAAAGACA AAATGCAC GTTTATTA AAACTATA CCATCCAA GATTGGGA AGGTAATA AGTCTTCA

GTTTCCTA AATTATGC TCAACAGC TTAACAAA CATTTCAG ATTATGAA GATTTAGG AGTTTACA TAATAGAT

AATTACCG CTACAAAATCCCGATA GTCAAGCGCGTAATTT CTGATTATCGGTAAGT TTTTATAACAGACACG GTGCATAA

A

B

D

C

F

G

I

H

J

K

M

L

AATGCATA AAATGGGC AAATTACC TTTGAAGG AGTACTTT TTCAGAAA

TTACGTAT TTTACCCG TTTAATGG AAACTTCC TCATGAAA AAGTCTTT

TCTAATGA GAACTACT TCCAGTCG GTCGGATA CGCGGACC AGACATGT GGCAAGTA GACAGGAG AAAGTTTC AACCAGTC AAGCCAAG GGAATACT AACTGGCA GACGCGGA GCAAGGCC GATTCATT GTACCTCG TCCAGCGC

AGATTACT CTTGATGA AGGTCAGC CAGCCTAT GCGCCTGG TCTGTACA CCGTTCAT CTGTCCTC TTTCAAAG TTGGTCAG TTCGGTTC CCTTATGA TTGACCGT CTGCGCCT CGTTCCGG CTAAGTAA CATGGAGC AGGTCGCG

CTTAGAAA GATGGACA TTATTACA ACAAGGCA ATCAAGCA AAATAATT GCATCTAA AAAGAAGG GTTGCAGG ACTGACCA TATTACTC GGTCAAGA ATTTTAGC GTATTCCA

GAATCTTT CTACCTGT AATAATGT TGTTCCGT TAGTTCGT TTTATTAA CGTAGATT TTTCTTCC CAACGTCC TGACTGGT ATAATGAG CCAGTTCT TAAAATCG CATAAGGT

TTATAAAT ACTGCTAA GGCGTCAT AACCTGCG ATAGGTCA GATTTGTA AAATGATA ATGGGGGA GACCGTTT TGAAGAAA ACGTTTTC GGAGAGCG ATAAAACC AAAAATAG

AATATTTA TGACGATT CCGCAGTA TTGGACGC TATCCAGT CTAAACAT TTTACTAT TACCCCCT CTGGCAAA ACTTCTTT TGCAAAAG CCTCTCGC TATTTTGG TTTTTATC

GCGTTTTT ACTGGAGA ATAGTTTT CCTCGTTA ATTTCCAT GAGAGATT AGGACTGG ACAACCTC AAACGAAG GCCAGACC AAGCGAAA CTTCGAGC TTAATTTT GCGCTATA AACTTCAG AAAGCCCG AAGGAGAA TTAGAAAA

CGCAAAAA TGACCTCT TATCAAAA GGAGCAAT TAAAGGTA CTCTCTAA TCCTGACC TGTTGGAG TTTGCTTC CGGTCTGG TTCGCTTT GAAGCTCG AATTAAAA CGCGATAT TTGAAGTC TTTCGGGC TTCCTCTT AATCTTTT

ATTACCAG TTTGATTTAGATGAGC AAGCGTCTTAACCCTT AGTTGACAATATACCT TACTTTGAAGGTCTGT GGCATGAAATCAACGT ATAAATTTTGTACAAC TCGATGTCGTAATATA AGTCGTTAATTCGAGA TTCGGTAG

TAATGGTC AAACTAAA TCTACTCG TTCGCAGA ATTGGGAA TCAACTGT TATATGGA ATGAAACT TCCAGACA CCGTACTT TAGTTGCA TATTTAAA ACATGTTG AGCTACAGCATTATAT TCAGCAAT TAAGCTCT AAGCCATC

GAGTCCGT AACGTAAATTTTATAT ACTCCCAAGATTTTTA AAAATAGGAACGCAAC TTTATTTCCGAAGAGG GCGTTTTCATAATGTC CCAGTATTACAAAAAC CATGTTGGCTAAATCG AAATACGAGACTCCGA AATAACGA

CTCAGGCA TTGCATTT AAAATATA TGAGGGTT CTAAAAAT TTTTATCC TTGCGTTG AAATAAAG GCTTCTCC CGCAAAAG TATTACAG GGTCATAA TGTTTTTG GTACAACCGATTTAGC TTTATGCT CTGAGGCT TTATTGCT

TTGCAAAT GTTAAATTTATAAACG AATATGTTAGAAGGAC AAAAACCCCGAAAAGA CTAATAGTTGGCCCCA TGTATACTAACTGTAC GATCAAAATGCTAATG GCAAGTAGCTAAGAGA ACAAACGAGGTCTGAG AGTCCGTT

AACGTTTA CAATTTAA ATATTTGC TTATACAA TCTTCCTG TTTTTGGG GCTTTTCT GATTATCA ACCGGGGT ACATATGA TTGACATG CTAGTTTT ACGATTAC CGTTCATCGATTCTCT TGTTTGCT CCAGACTC TCAGGCAA

TTTCGACC GACCTCACGCTAGAAG GACTCCGGCTATGACA GCAGCAGGGGAGTTTG ACCGTCTACGTGCCAA TGCTACGCGGGTAGAT GTGGTTGCACTGGATA GGGTAATGCCAGTTAG GCGGCAAACAAGGGTG CCTCTTAG

AAAGCTGG CTGGAGTG CGATCTTC CTGAGGCC GATACTGT CGTCGTCC CCTCAAAC TGGCAGAT GCACGGTT ACGATGCG CCCATCTA CACCAACG TGACCTAT CCCATTACGGTCAATC CGCCGTTT GTTCCCAC GGAGAATC

TTGAATTA GCGGAACG TCGTGTAG GGGGAAAG CGGTCGAC CGCATTAT CGCTTCTC CGGGCGTG GCTAGCGG GAAGGGTT GTCAACGC GTCGGACT TACCGCTT ACCGCGAA ACGGACCA AAGGCCGT GGTCTTCG CCACGGCC

AACTTAAT CGCCTTGC AGCACATC CCCCTTTC GCCAGCTG GCGTAATA GCGAAGAG GCCCGCAC CGATCGCC CTTCCCAA CAGTTGCG CAGCCTGA ATGGCGAA TGGCGCTT TGCCTGGT TTCCGGCA CCAGAAGC GGTGCCGG

GGACGACC CCGTTTGG TCGCACCT GGCGAACG ACGTTGAG AGAGTCCC GGTCCGCC ACTTCCCG TTAGTCGA CAACGGGC AGAGTGAC

CCTGCTGG GGCAAACC AGCGTGGA CCGCTTGC TGCAACTC TCTCAGGG CCAGGCGG TGAAGGGC AATCAGCT GTTGCCCG TCTCACTG

AAGCGGGA AACTGCAACCTCAGGT GCAAGAAATTATCACC TGAGAACAAGGTTTGA CCTTGTTGTGAGTTGG GATAGAGCCCGATAAG AAAACTAAATATTCCC TAAAACGGCTAAAGCC TTGGTGGTAGTTTGTC CTAAAAGC

TTCGCCCT TTGACGTT GGAGTCCA CGTTCTTT AATAGTGG ACTCTTGT TCCAAACT GGAACAAC ACTCAACC CTATCTCG GGCTATTC TTTTGATT TATAAGGG ATTTTGCCGATTTCGG AACCACCA TCAAACAG GATTTTCG

TACGATGT CCGCAACATCAAACAT GACCACTGCTTTGAGT CACAATGCCATGTACC CAAGGATAACCCGAACGATAGGGA

GGCCCGAT ATGAATAT AGTTGGGA GAGCTGCC GTGAATAG GCGGACCA TGACTCGT TTTGGGGC GATTAGGA TTAGGAAG AGAACTCC TCAGAGTC GGAGAATT ATGAAAGT ACAAAGTC TTATTATC CAAGGCTT TATCCGTC

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

CCCCGTAA TTGACAAA TATGCCCG TGACAATG AGTTCCGT GACTGGGG CAATTTTG AATAATGGTCATGTGA GGACATAGTAGTTTTC GGTACATA CTGCGAAT GACCTTGC CATTTAAG TCTCTGAC GCGAAAGG TAAGACCG

GGGGCATT AACTGTTT ATACGGGC ACTGTTAC TCAAGGCA CTGACCCC GTTAAAAC TTATTACC AGTACACT CCTGTATC ATCAAAAG CCATGTAT GACGCTTA CTGGAACGGTAAATTC AGAGACTG CGCTTTCC ATTCTGGC

CCGCCGAG ACTCCCTC CGCCAAGG CCACCACC GAGACCAA GGCCACTA AAACTAAT ACTTTTCT ACCGTTTG CGATTATT CCCCCGAT ACTGGCTT TTACGGCT ACTTTTGC GCGATGTC AGACTGCG ATTTCCGT TTGAACTA

GGCGGCTC TGAGGGAG GCGGTTCC GGTGGTGG CTCTGGTT CCGGTGATTTTGATTA TGAAAAGA TGGCAAAC GCTAATAA GGGGGCTA TGACCGAA AATGCCGA TGAAAACG CGCTACAG TCTGACGC TAAAGGCA AACTTGAT

AGCAGACCTGTCTTAATGAAATGG AAAACAGCCATGAAAT ATAAGAGAATAATGAC CGAGCTTTTACGGAGA CGGATTTAATGTACAA CCGCAACAATTTATAC CGCTAAGAGTTAATTC GGGATGACAACTCGCA ACCGAAAT

TCGTCTGG ACAGAATT ACTTTACC TTTTGTCG GTACTTTA TATTCTCT TATTACTG GCTCGAAA ATGCCTCT GCCTAAAT TACATGTT GGCGTTGT TAAATATG GCGATTCTCAATTAAG CCCTACTG TTGAGCGT TGGCTTTA

TTGATTTT ATATAAACTTTTTCAA AAGAGCGCAAGAAACA GAACGCTAACCTAAAC GTAGTCGTAAATGTAT ATCAATATATTGGGTT GGATTCGGCCTCCAAT TTTTCCATCAGAGAGT CTGGATACTAAAACTA TTTAAGTG

AACTAAAA TATATTTG AAAAAGTT TTCTCGCG TTCTTTGT CTTGCGAT TGGATTTG CATCAGCA TTTACATA TAGTTATA TAACCCAA CCTAAGCC GGAGGTTA AAAAGGTAGTCTCTCA GACCTATG ATTTTGAT AAATTCAC

ATTAAGTTTACTTTAA CAATTTAC ATTAATTAAAACAAAA GAACTACAAACAAAGT AGTAGAAGAAAACGAG TCCATTAACTTTACTT ATTAAGCGGAGACGCG CTAAAACATTGAACCA TAAGTTTCGTTAGTCC GCTTAGGC

TAATTCAA ATGAAATT GTTAAATG TAATTAAT TTTGTTTT CTTGATGT TTGTTTCA TCATCTTC TTTTGCTC AGGTAATT GAAATGAA TAATTCGC CTCTGCGC GATTTTGTAACTTGGT ATTCAAAG CAATCAGG CGAATCCG

TATTAGGT TTGTTAGTCCTAATAT AACTACTTAACGGTAG TAGACTATTAGTCCTT ATACTACTATTAAGGC GAGGAAGACCACCAAA GAAACAAGGCGTTTTA CTATTACAATGAGTTT GAAAATTTTAATTATT GCAAGCCC

ATAATCCA AACAATCA GGATTATA TTGATGAA TTGCCATC ATCTGATA ATCAGGAA TATGATGA TAATTCCG CTCCTTCT GGTGGTTT CTTTGTTC CGCAAAAT GATAATGTTACTCAAA CTTTTAAA ATTAATAA CGTTCGGG

GACAACGT CCGCCACA ATTATGAC TGGCGGAG TGGAGACA AAATAGAA GACGACCA CCAAGCAA GCCATAAA

CTGTTGCA GGCGGTGT TAATACTG ACCGCCTC ACCTCTGT TTTATCTT CTGCTGGT GGTTCGTT CGGTATTT

GAATGCGA AAGTCCAG TCTTCCCA AGATAGAG ACAACCGG TCTTACAG GGAAAATA ATGACCAG CACACTGA CCACTTAGCTGGGGTT TTTTGAACTAAACCCA CTACCAAGTGCATCAC CCGGTAGCGGGACTAT CTGCCAAA

GACCCCAA AAAACTTG ATTTGGGT GATGGTTC ACGTAGTG GGCCATCG CCCTGATA GACGGTTT

TGTCATTG TCGGCGCA ACTATCGG TATCAAGC TGTTTAAG AAATTCAC - CTCGAAAG CAAGCTGA TAAACCGA TACAATTA AAGGCTCC TTTTGGAG CCTTTTTT TTGGAGAT TTTCAACG TGAAAAAA

AAAGCGAC GACTCCCA CTGCTAGG GCGTTTTC GCCGGAAA TTGAGGGA

C

D

I

H

J

K

L

N

CGTTCGGA GTCGCTGGCTTATATA GCCAATACGCACCCGC TACCAACA

GCAAGCCT CAGCGACC GAATATAT CGGTTATG CGTGGGCG ATGGTTGT

AATAATAA GCGTTAAGGAAATCAA CAAGGAAAGATAAGAG TGAGGCGACTTTGACA ACTTTCAACAAATCGT TTTAGGGT

TTATTATT CGCAATTC CTTTAGTT GTTCCTTT CTATTCTC ACTCCGCT GAAACTGT TGAAAGTT GTTTAGCA AAATCCCA

ATGTCTTT TAAGTAAA TGATTGCA GACCTTTC TGCTGTTT TGAAATCT AGCAATGC GATTGATA CTCCCGAC AGACACCT

TACAGAAA ATTCATTT ACTAACGT CTGGAAAG ACGACAAA ACTTTAGA TCGTTACG CTAACTAT GAGGGCTG TCTGTGGA

CTTTTACT CCCACCAC CGAGACTC CCACCGCC AAGACTCC CACCGCCA AGACTCCC ACCGCCAT GATTTGGA GGACTCAT GCCACTAT GTGGATAA

ATGCTACA GGCGTTGT AGTTTGTA CTGGTGAC GAAACTCA GTGTTACG GTACATGG GTTCCTAT TGGGCTTG CTATCCCT

TGCTCCTT TCGTGCAA TATGCACG AGCAGTTT CGTTGGTA TCATGCGC GGGACATC GCCGCGTA ATTCGCGC CGCCCACA

ACGAGGAA AGCACGTT ATACGTGC TCGTCAAA GCAACCAT AGTACGCG CCCTGTAG CGGCGCAT TAAGCGCG GCGGGTGT

GACCGCCA TTATAACA AGACCTAT AATGGTCG TTCCGGCT ATCAAACT CAAGAAGA TGAGTCCG TTCACTAC AATAATGA

CTGGCGGT AATATTGT TCTGGATA TTACCAGC AAGGCCGA TAGTTTGA GTTCTTCT ACTCAGGC AAGTGATG TTATTACT

GGTGGTTA CGCGCAGC GTGACCGC TACACTTG CCAGCGCC CTAGCGCC CGCTCCTT TCGCTTTC TTCCCTTC CTTTCTCG

CCACCAAT GCGCGTCGCACTGGCG ATGTGAACGGTCGCGG GATCGCGGGCGAGGAA AGCGAAAGAAGGGAAG GAAAGAGC

CCACGTTC GCCGGCTT TCCCCGTC AAGCTCTA AATCGGGG GCTCCCTT TAGGGTTC CGATTTAG TGCTTTAC GGCACCTC

GGTGCAAG CGGCCGAA AGGGGCAG TTCGAGAT TTAGCCCC CGAGGGAA ATCCCAAG GCTAAATC ACGAAATG CCGTGGAG

ACGGTTAC ATTTATTAGGTAAAGT CTGCTAACTCGCAGTT TTACATCCATAAAGGT ACTCGCAAAAAGGACA ACGTTACC

TGCCAATG TAAATAAT CCATTTCA GACGATTG AGCGTCAA AATGTAGG TATTTCCA TGAGCGTT TTTCCTGT TGCAATGG

E

9 10 11 12 13 14 15 16 17 18

TTGACGGC TCTAATCT ATTAGTTG TTAGTGCT CCTAAAGATATT

AACTGCCG AGATTAGA TAATCAAC AATCACGA

TTAGATAACCTT CCTCAATT CCTTTCAA CTGTTGAT TTGCCAAC -- TGACCAGA TATTGATT GAGGGTTT GATATTTG AGGTTCAGCAAGG

GGAGTTAA GGAAAGTT GACAACTA AACGGTTG ACTGGTCT ATAACTAA CTCCCAAA CTATAAAC

TGATGCTTTAGA TTTTTCAT TTGCTGCT GGCTCTCA GCGTGGCA

AAAAAGTA AACGACGA CCGAGAGT CGCACCGT

MR

CTTACGCT TTCAGGTC AGAAGGGT TCTATCTC TGTTGGCC AGAATGTC CCTTTTAT TACTGGTC GTGTGACT GGTGAATC

M

CGCAATTA ATGTGAGT TAGCTCAC TCATTAGG CACCCCAG

CCTCTCCC CGCGCGTT GGCCGATT CATTAATG CAGCTGGC -- ACGACAGG TTTCCCGA CTGGAAAG CGGGCAGT GAGCGCAA

TACACTCA ATCGAGTG AGTAATCC GTGGGGTC

GCGCGCAA CCGGCTAA GTAATTAC GTCGACCG TGCTGTCC AAAGGGCT GACCTTTC GCCCGTCA

CACTTTTC TTTTTGGT GGGACCGC GGGTTATG

GTGAAAAG AAAAACCA CCCTGGCG CCCAATAC GCAAACCG

ML

N

TCTGTCGC TACTGATT ACGGTGCT GCTATCGA TGGTTTCA TTGGTGAC GTTTCCGG CCTTGCTA ATGGTAAT GGTGCTAC TGGTGATT TTGCTGGC TCTAATTC CCAAATGG CTCAAGTC GGTGACGG TGATAATT CACCTTTA ATGAATAA TTTCCGTC AATATTTA CCTTCCCT

CCCTCAAT CGGTTGAA TGTCGCCC TTTTGTCT TTGGCGCT GGTAAACC ATATGAAT TTTCTATT GATTGTGA CAAAATAA ACTTATTC CGTGGTGT CTTTGCGT TTCTTTTATATGTTGC CACCTTTA TGTATGTA TTTTCTAC GTTTGCTA ACATACTG CGTAATAA GGAGTCTT

TTATTTTG TAACTGGC AAATTAGG CTCTGGAA AGACGCTC GTTAGCGT TGGTAAGA TTCAGGAT AAAATTGT AGCTGGGT GCAAAATA GCAACTAA TCTTGATT TAAGGCTT CAAAACCT CCCGCAAG TCGGGAGG TTCGCTAA

AGACAGCG ATGACTAA TGCCACGA CGATAGCT ACCAAAGT AACCACTG CAAAGGCC GGAACGAT TACCATTA CCACGATG ACCACTAA AACGACCG AGATTAAG GGTTTACC GAGTTCAG CCACTGCC ACTATTAA GTGGAAAT TACTTATT AAAGGCAG TTATAAAT GGAAGGGA

GGGAGTTA GCCAACTTACAGCGGG AAAACAGAAACCGCGA CCATTTGGTATACTTA AAAGATAACTAACACT GTTTTATTTGAATAAG GCACCACAGAAACGCA AAGAAAATATACAACG GTGGAAATACATACATAAAAGATGCAAACGAT TGTATGACGCATTATTCCTCAGAA

AATAAAAC ATTGACCG TTTAATCC GAGACCTT TCTGCGAG CAATCGCA ACCATTCT AAGTCCTA TTTTAACA TCGACCCA CGTTTTAT CGTTGATT AGAACTAA ATTCCGAA GTTTTGGA GGGCGTTC AGCCCTCC AAGCGATT

TTAGTACG GTCAAGAA AACCCATA AGGCAATA ATAACGCA AAGGAGCC AAAGGAAG ACCATTGA AACAAGCC GATAGACG AATGAAAA GAATTTTT CCCGAAGC CATTCTAT CGATAACG ATAAAGTA ACAAAGAA CGAGAATA ATAACCCG AATTGAGT TAAGAACA CCCAATAG

AATCATGC CAGTTCTT TTGGGTAT TCCGTTAT TATTGCGT TTCCTCGG TTTCCTTC TGGTAACT TTGTTCGG CTATCTGC TTACTTTT CTTAAAAA GGGCTTCG GTAAGATA GCTATTGC TATTTCAT TGTTTCTT GCTCTTAT TATTGGGC TTAACTCA ATTCTTGT GGGTTATC

AGAGACTA TAATCGCGAGTTAATG GGAGACTGAAACAAGT CCCACAAGTCAATTAA GAGGGCAGATTACGCG AAGGGACAAAAATACA ATAAGAGAGACATTTC CGACGATAAAAGTAAA AACTGCAATTTGTTTT TTAGCAAAGAATAAAC CTAACCCTATTTATTA TACCGACA

TCTCTGAT ATTAGCGC TCAATTAC CCTCTGAC TTTGTTCA GGGTGTTC AGTTAATT CTCCCGTC TAATGCGC TTCCCTGT TTTTATGT TATTCTCT CTGTAAAG GCTGCTATTTTCATTT TTGACGTT AAACAAAA AATCGTTT CTTATTTG GATTGGGA TAAATAAT ATGGCTGT

E

F

AACGCCTC GCGTTCTT AGAATACC GGATAAGC CTTCTATA TCTGATTT GCTTGCTA TTGGGCGC GGTAATGA TTCCTACG ATGAAAAT AAAAACGG CTTGCTTG TTCTCGATGAGTGCGG TACTTGGT TTAATACC CGTTCTTG

TTGCGGAG CGCAAGAA TCTTATGG CCTATTCGGAAGATAT AGACTAAACGAACGAT AACCCGCGCCATTACT AAGGATGCTACTTTTA TTTTTGCCGAACGAAC AAGAGCTACTCACGCC ATGAACCAAATTATGG GCAAGAAC G

TTTTTTTT

TTTTTTTT

TTTTTTTT

TTTTTTTT

JP- 1Staple strands indicated by bold lines are hairpin-incorporated strands.

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).

Lock M13mp18 and release using PVS

i) Mix M13mp18 + Buffer + 8K90Dii) Incubate at room temp for 30 miniii) Add Staple strandsiv) Add PVS / For control don’t add PVSv) Anneal 85-15°C (-1°C/min)vi) Purify using S-500vii) AFM imaging

1400 × 1050 nm

Polymer : M13 = 1 : 1

N/P = 0.2

1500 × 1125 nm

Polymer : M13 = 1.5 : 1

N/P = 0.3

1400 × 1050 nm

2480 × 1860 nm

2400 × 1800 nm

Polymer : M13 = 2 : 1

N/P = 0.4

2400 × 1800 nm

2400 × 1800 nm

Polymer : M13 = 4 : 1

N/P = 0.8

2400 × 1800 nm

N/P = 0.1 1500 × 1125 nm

Polymer : M13 = 0.5 : 1

1500 × 1125 nm

2480 × 1860 nmN/P = 0.25

Polymer : M13 = 1.25 : 1

1800 × 1350 nm

2000 × 1500 nmN/P = 0.6

Polymer : M13 = 3 : 1

1000 × 750 nm

PV

S a

dded

No

PV

S

PV

S a

dded

No

PV

S

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 staple strands and release using PVS

i) Mix Staples + Buffer + 8K90Dii) Incubate at room temp for 30 miniii) Add M13mp18iv) Add PVS / For control don’t add PVSv) Anneal 85-15°C (-1°C/min)vi) Purify using S-500vii) AFM imaging

2380 × 1785 nm

Polymer : Staples = 1 : 1

N/P = 0.8

1400 × 1050 nm

2480 × 1860 nm

Polymer : Staples = 1.5 : 1

N/P = 1.2

2480 × 1860 nm 2480 × 1860 nm

2480 × 1860 nm

Polymer : Staples = 2 : 1

N/P = 1.6

1600 × 1200 nm

Polymer : Staples = 0.25 : 1

N/P = 0.2

2000 × 1500 nm

1200 × 900 nm

Polymer : Staples = 0.5 : 1

N/P = 0.4

2480 × 1860 nm

PV

S a

dded

No

PV

S

PV

S a

dded

No

PV

S

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 both M13mp18 and staple strands at the same time and release using PVS

i) Mix M13mp18 + Staples + Buffer + 8K90Dii) Incubate at room temp for 30 miniii) Add PVS / For control don’t add PVSiv) Anneal 85-15°C (-1°C/min)v) Purify using S-500vi) AFM imaging

1500 × 1125 nm

Polymer : (M13 + Staples) = 0.25 : 1

N/P = 0.25

1200 × 900 nm

Polymer : (M13 + Staples) = 0.25 : 12400 × 1800 nm

Polymer : (M13 + Staples) = 0.5 : 1

N/P = 0.5

2000 × 1500 nm

Polymer : (M13 + Staples) = 0.5 : 11600 × 1200 nm

Polymer : (M13 + Staples) = 1 : 1

N/P = 1

1200 × 900 nm

Polymer : (M13 + Staples) = 1 : 1

PV

S a

dded

No

PV

S

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.

Lock M13Polymer : M13 = 5 : 1

Lock staplesPolymer : Staples = 1.25 : 1

Lock both M13 and staplesPolymer : (M13 + Staples) = 1 : 1

Table S1. The yield of the jigsaw-shaped origami structure at different conditions

Polymer Lock Polymer:DNA N/P Ratio Yield Number of Tiles Calculated 8K90D M13 2:1 0.4 90% 202 8K90D Staples 0.5:1 0.4 86% 165 8K90D Both 0.5:1 0.5 72% 79 15K92D M13 5:1 1 95% 144 15K92D Staples 1.25:1 1 90% 208 15K92D Both 1:1 1 86% 234

All experimental conditions are same as given in main text Fig. 2 and 3.

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).

Simultaneous addition of polymer and PVS

i) Mix M13mp18 + Staples + Buffer + 8K90D/15K92D + PVSii) Incubate at room temp for 30 miniii) Anneal 85-15°C (-1°C/min)iv) Purify using S-500v) AFM imaging

2480 × 1860 nm

8K90D : (M13 + Staples) = 1 : 1

N/P = 1 1200 × 900 nm

15K92D : (M13 + Staples) = 1 : 1

N/P = 1

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 M13 Lock staples Lock both M13 and staples

1400 × 1050 nm

Polymer : M13 = 5 : 1

N/P = 1 2480 × 1860 nm

Polymer : Staples = 1.25 : 1

N/P = 1 2480 × 1860 nm

Polymer : (M13 + Staples) = 1 : 1

N/P = 1

2480 × 1860 nm 2480 × 1860 nm 2480 × 1860 nm

No

PV

S

No

PV

S

40 m

MPh

osph

ate

buffe

rPo

lym

er :

Phos

phat

e Bu

f= 1

: 11

020

mM

Phos

phat

e bu

ffer

Poly

mer

: Ph

osph

ate

Buf=

1 :

55

i) Mix M13mp18 + Phosphate buffer + 8K90Dii) Incubate at room temp for 30 miniii) Add staple strandsiv) Anneal 85-15°C (-1°C/min)v) Purify using S-500vi) 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).

Lock M13 Lock staples Lock both M13 and staples

2480 × 1860 nm

Polymer : M13 = 5 : 1

N/P = 1 2480 × 1860 nm

Polymer : Staples = 1.25 : 1

N/P = 1 2480 × 1860 nm

Polymer : (M13 + Staples) = 1 : 1

N/P = 1

2480 × 1860 nm 2480 × 1860 nm 2480 × 1860 nm

No

PV

S

No

PV

S

2 m

MN

TPs

Pol

ymer

: N

TPs

= 1

: 16.

55.

64 m

MN

TPs

Pol

ymer

: N

TPs

= 1

: 46.

6

i) Mix M13mp18 + Buffer + NTPs + 8K90Dii) Incubate at room temp for 30 miniii) Add staple strandsiv) Anneal 85-15°C (-1°C/min)v) Purify using S-500vi) AFM imaging