SUPPLEMENTARY INFORMATION...origami tiles was mixed with 4 µL each of a 50 nm solutions containing cassettes 1 and 2. The system ... then the solution was left at 25 ˚C for 2 hours

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nnano.2009.5

nature nanotechnology | www.nature.com/naturenanotechnology 1

1

Dynamic Patterning Programmed by DNA Tiles Captured on a DNA

Origami Substrate

Hongzhou Gu, Jie Chao, Shou-Jun Xiao & Nadrian C. Seeman*

SUPPLEMENTARY MATERIAL.

The supplementary material contains the experimental methods, sequences of origami staple strands, the

design of the origami (Figure S1), the sequences of the capture molecules (Figures S2-S5), the

sequences of the cassettes (Figure S6), nondenaturing gels of the capture molecules, and cassettes and

their combination (Figure S7), and the correction procedure applied to the three systems not shown in

the main text, the linear system (Figure S8), the triangle pointing away from the notch (Figure S9) and

the triangle pointing towards the notch (S10). Figure S11 shows the results of omitting the first step of

the correction procedure. Table S1 shows quantitation of the correction procedure. References are at

the end.

Experimental Methods

Design, Synthesis and Purification of DNA. Strands for the cassettes and capture target molecules

have been designed using the program SEQUIN (1). Oligonucleotides have been synthesized on an

Applied Biosystems 394 synthesizer using routine phosphoramidite chemistry or have been purchased

from the Integrated DNA Technology (www.idtDNA.com). DNA strands have been purified by gel

electrophoresis: bands are cut out of 10-20% denaturing gels and eluted in a solution containing 500

mM ammonium acetate, 10 mM magnesium acetate, and 1 mM EDTA.

Formation of Hydrogen-Bonded DNA Device and Target Molecule Complexes: Stoichiometric

mixtures of the strands (estimated by OD260) were prepared separately for each tile to a concentration of

0.05 �� in a solution containing 40 mM Tris-HCl, pH 8.0, 20 mM acetic acid, 2.5 mM EDTA, and 12.5

mM magnesium acetate. Each mixture was cooled from 90 ˚C to room temperature in a 500-mL water

bath over the course of 48 h.

© 2009 Macmillan Publishers Limited. All rights reserved.

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SUPPLEMENTARY INFORMATION doi: 10.1038/nnano.2009.5

2

Formation of the DNA origami tiles: 5 µL of 0.03 �M (= 0.15 pmol) M13 plasmid (New England

Biolabs) was combined with the staple strands (1:7 molar ratio of plasmid to staple strands) to a buffer

solution containing 40 mM Tris-HCl, pH 8.0, 20 mM acetic acid, 2.5 mM EDTA, and 12.5 mM

magnesium acetate. The final volume for the system was 100 µL. The system was cooled from 90 ˚C to

60 ˚C on a thermocycling machine over 30 m, and then cooled further to 16 ˚C over 90 m. The

concentration of the DNA origami at this stage was 1.5 nM. Insofar as could be estimated by AFM, the

yield was virtually quantitative.

Purification of the DNA origami: The procedure of Ke et al (2). was used to remove excess helper

strands from the origami solution. The origami tiles were purified with Micro-con centrifugal filter

devices [MWCO 50,000 (Millipore, Bedford, MA)]. We adjusted the final concentration of the origami

tiles to 1 nM estimated by OD260.

Insertion of the Two Cassettes into the DNA Origami: 200 µL of a solution containing 1 nM

origami tiles was mixed with 4 µL each of a 50 nm solutions containing cassettes 1 and 2. The system

was heated to 45 ˚C and slowly cooled to 4 ˚C over 1 day in a 2 L water bath. The final solution

contains 0.2 pmole of each of the three components in a 1:1:1 molar ratio. Insofar as could be estimated

by AFM, the yield of capturing one cassette was virtually quantitative and the yield of capturing both

cassettes was 90-95%.

Operation of Device on the Origami: The PX state was the default state when the two cassettes

were inserted into the Origami, so the system was initiated as with PX(cassette-1)/PX(cassette-2). To

switch to a different state, e.g., PX(cassette-1)/JX2(cassette-2), 4.2 µL each of 50 nM solutions

containing strands Fuel-P1 and Fuel-P2 for cassette-2 was added to the above 208 µL solution (Origami:

cassette-1: cassette-2 = 1:1:1, each at a concentration of 1 nM). The solution was stirred with a pipette

for 5 min, then the solution was left at 25 ˚C for 2 hours. The mixture was then treated with streptavidin

beads at 25 ˚C for 45 min. The tube with the mixture solution was then put on a magnetic stand for

another 45 min to allow beads with the set-strand/fuel-strand duplexes to gather at the bottom. The

mixture solution was then transferred to a new tube. 4.2 µL each of 50 nM solutions containing Set-J1

and Set-J2 strands for cassette-2 was added to the solution. The solution was stirred again with a pipette

for 5 min then kept at 25 ˚C for 6 hours to establish the new PX(cassette-1)/JX2(cassette-2)

conformation before doing corrections. Note that the use of magnetic bead technology precludes the

3

back conversion of the JX2(cassette-2) to PX(cassette-2) by removing the PX set strands. The two

different ways of setting the state of the system, programming the cassettes before binding to the

origami or inserting the cassettes in a default state into the origami and then reprogramming, produced

the same results.

Correction Procedure: After the insertion of cassettes, 200 µL of the origami system is treated with

a mixture of all four capture molecules (1 µL each of a 50 nM�solution). The solution is then heated up

to 37 – 40 ˚C and cooled down slowly to 4 ˚C over 1 day in a 2 L water bath. The remaining solution is

treated with one of the four species (4 µL of a 50 nM solution) and the thermal protocol is repeated.

This procedure is repeated until all of the four species have been added in this fashion.

Non-denaturing Polyacrylamide Gel Electrophoresis. Non-denaturing gels contain of 4%

acrylamide (19:1, acrylamide: bisacrylamide) and the running buffer contains of 40 mM Tris-HCl (pH

8.0), 20 mM acetate acid, 2 mM EDTA, and 12.5 mM magnesium acetate (1XTAE/Mg). Tracking dye

containing 1XTAE/Mg, 50% glycerol, and 0.02% each of Bromophenol Blue and Xylene Cyanol FF is

added to the sample buffer. Gels are run on a Hoefer SE-600 gel electrophoresis unit at 4V/cm at 4ºC

and were stained with 0.01% Stains-all dye (Sigma), in 45% formamide.

Atomic Force Microscopy (AFM) Imaging. A 5 µL sample was spotted on freshly cleaved mica,

and the sample was left to absorb for 2 minutes. Additional fresh 1XTAE/Mg (12.5mM) buffer was

added to both the mica and to the liquid cell. All AFM imaging was performed on a NanoScope IV

(Digital Instruments), using commercial cantilevers with Si3N4 tips.

Sequences of Origami Helper Strands

Referring to Figure S1, the sequence is named as X, Y.

X refers to the column, which is counted from left to right.

Y refers to the row, which is counted from bottom to top.

1, 1; GGCCGATTAAAGGGATGAGAGGCG

1, 2; GTTTGCGTTTTTTGAAATTGTTATCCGCATAGCTGT

1, 3; TTCCTGTGTTTTCTTCGCTATTACGCCACGATCGGT

1, 4; GCGGGCCTTTTTAGCTTTCCGGCACCGCAGATCGCA




2

Formation of the DNA origami tiles: 5 µL of 0.03 �M (= 0.15 pmol) M13 plasmid (New England

Biolabs) was combined with the staple strands (1:7 molar ratio of plasmid to staple strands) to a buffer

solution containing 40 mM Tris-HCl, pH 8.0, 20 mM acetic acid, 2.5 mM EDTA, and 12.5 mM

magnesium acetate. The final volume for the system was 100 µL. The system was cooled from 90 ˚C to

60 ˚C on a thermocycling machine over 30 m, and then cooled further to 16 ˚C over 90 m. The

concentration of the DNA origami at this stage was 1.5 nM. Insofar as could be estimated by AFM, the

yield was virtually quantitative.

Purification of the DNA origami: The procedure of Ke et al (2). was used to remove excess helper

strands from the origami solution. The origami tiles were purified with Micro-con centrifugal filter

devices [MWCO 50,000 (Millipore, Bedford, MA)]. We adjusted the final concentration of the origami

tiles to 1 nM estimated by OD260.

Insertion of the Two Cassettes into the DNA Origami: 200 µL of a solution containing 1 nM

origami tiles was mixed with 4 µL each of a 50 nm solutions containing cassettes 1 and 2. The system

was heated to 45 ˚C and slowly cooled to 4 ˚C over 1 day in a 2 L water bath. The final solution

contains 0.2 pmole of each of the three components in a 1:1:1 molar ratio. Insofar as could be estimated

by AFM, the yield of capturing one cassette was virtually quantitative and the yield of capturing both

cassettes was 90-95%.

Operation of Device on the Origami: The PX state was the default state when the two cassettes

were inserted into the Origami, so the system was initiated as with PX(cassette-1)/PX(cassette-2). To

switch to a different state, e.g., PX(cassette-1)/JX2(cassette-2), 4.2 µL each of 50 nM solutions

containing strands Fuel-P1 and Fuel-P2 for cassette-2 was added to the above 208 µL solution (Origami:

cassette-1: cassette-2 = 1:1:1, each at a concentration of 1 nM). The solution was stirred with a pipette

for 5 min, then the solution was left at 25 ˚C for 2 hours. The mixture was then treated with streptavidin

beads at 25 ˚C for 45 min. The tube with the mixture solution was then put on a magnetic stand for

another 45 min to allow beads with the set-strand/fuel-strand duplexes to gather at the bottom. The

mixture solution was then transferred to a new tube. 4.2 µL each of 50 nM solutions containing Set-J1

and Set-J2 strands for cassette-2 was added to the solution. The solution was stirred again with a pipette

for 5 min then kept at 25 ˚C for 6 hours to establish the new PX(cassette-1)/JX2(cassette-2)

conformation before doing corrections. Note that the use of magnetic bead technology precludes the

3

back conversion of the JX2(cassette-2) to PX(cassette-2) by removing the PX set strands. The two

different ways of setting the state of the system, programming the cassettes before binding to the

origami or inserting the cassettes in a default state into the origami and then reprogramming, produced

the same results.

Correction Procedure: After the insertion of cassettes, 200 µL of the origami system is treated with

a mixture of all four capture molecules (1 µL each of a 50 nM�solution). The solution is then heated up

to 37 – 40 ˚C and cooled down slowly to 4 ˚C over 1 day in a 2 L water bath. The remaining solution is

treated with one of the four species (4 µL of a 50 nM solution) and the thermal protocol is repeated.

This procedure is repeated until all of the four species have been added in this fashion.

Non-denaturing Polyacrylamide Gel Electrophoresis. Non-denaturing gels contain of 4%

acrylamide (19:1, acrylamide: bisacrylamide) and the running buffer contains of 40 mM Tris-HCl (pH

8.0), 20 mM acetate acid, 2 mM EDTA, and 12.5 mM magnesium acetate (1XTAE/Mg). Tracking dye

containing 1XTAE/Mg, 50% glycerol, and 0.02% each of Bromophenol Blue and Xylene Cyanol FF is

added to the sample buffer. Gels are run on a Hoefer SE-600 gel electrophoresis unit at 4V/cm at 4ºC

and were stained with 0.01% Stains-all dye (Sigma), in 45% formamide.

Atomic Force Microscopy (AFM) Imaging. A 5 µL sample was spotted on freshly cleaved mica,

and the sample was left to absorb for 2 minutes. Additional fresh 1XTAE/Mg (12.5mM) buffer was

added to both the mica and to the liquid cell. All AFM imaging was performed on a NanoScope IV

(Digital Instruments), using commercial cantilevers with Si3N4 tips.

Sequences of Origami Helper Strands

Referring to Figure S1, the sequence is named as X, Y.

X refers to the column, which is counted from left to right.

Y refers to the row, which is counted from bottom to top.

1, 1; GGCCGATTAAAGGGATGAGAGGCG

1, 2; GTTTGCGTTTTTTGAAATTGTTATCCGCATAGCTGT

1, 3; TTCCTGTGTTTTCTTCGCTATTACGCCACGATCGGT

1, 4; GCGGGCCTTTTTAGCTTTCCGGCACCGCAGATCGCA




4

1, 5; CTCCAGCCTTTTAGGTTACGTTGGTGTATTGACCGT

1, 6; AATGGGATTTTTCTTCCTGTAGCCAGCTATAATTCG

1, 7; CGTCTGGCTTTTAACGTTAATATTTTGTAAATATTT

1, 8; AAATTGTATTTTAATCGTAAAACTAGCAAGAATCGA

1, 9; TGAACGGTTTTTAAACCATCGATAGCAGGGAAACGT

1, 10; CACCAATGTTTTATGAAAATAGCAGCCTTTGTTTAACGTCAAAA

2, 1; CCACACAATCGGCCAACGCGCGGGTTTAGACA

2, 2; AAGGGGGAATTCGTAATCATGGTCTCACAATT

2, 3; CCGGAAACCAACTGTTGGGAAGGGGCTGGCGA

2, 4; CATCGTAACGTATCGGCCTCAGGATTCTGGTG

2, 5; CATTAAATGGGAACAAACGGCGGAGATGGGCG

2, 6; GCGTTAAAAGGAACGCCATCAAAATTCATCAA

2, 7; ATATGTACGGAAGATTGTATAAGCTAAAATTC

2, 8; TCAGTAGCGTCTGGAGCAAACAAGTGTCAATC

2, 9; AGAATAACTACCATTAGCAAGGCCCACCGTAA

2, 10; CCAATCCAAATAAGAAACGATTTTTTACAGAG

3, 1; GGAACGGTACGCCAGAATCTTGAGCAGCTGCA

3, 2; TTAATGAACATACGAGCCGGAAGCCGGGTACC

3, 3; GAGCTCGATGTGCTGCAAGGCGATTAAGTTGG

3, 4; AGGCTGCGCAGGCAAAGCGCCATTTAATCTTC

3, 5; ACGACGACCCGTGCATCTGCCAGTTTCAACTA

3, 6; TTCTCCGTGTGAGCGAGTAACAACAGAAGCAA

3, 7; TCATTTTTTAACCAATTTTTTGTTAAATCAGC

3, 8; CAAAAACACCCGGTTGATAATCAGAAATTAAG

3, 9; GCCTGAGAGACAGAATCAAGTTTGTCACCAGT

3, 10; AGCACCATATAAAAACAGGGAAGCTATTTATC

5

4, 1; TAAAGCCTGGGAAACCTGTCGTGCAAGTGTTT

4, 2; GTAACGCCGACTCTAGAGGATCCCATAAAGTG

4, 3; GACAAGAATGACCTTCATCAAGAGCGCCATTC

4, 4; ATGCAGATATTTAGGAATACCACATTGAGGGG

4, 5; AGCGGATTCCTGACTATTATAGTCCCGTCGGA

4, 6; CAATAAAGAGGCAAAGAATTAGCAAAAAGCCC

4, 7; GTCAGACTATCTACAAAGGCTATCAGGTCATT

4, 8; CGGGAGAAATTAGAGCCAGCAAAACCTTTAGC

4, 9; CAGTTACAAAATAAACAGCCATATGCATTAGA

5, 1; TTATAATCAGTGAGGCCACCGAGTTGCCCGCT

5, 2; TTCCAGTCGGGGTGCCTAATGAGTTGCATGCC

5, 3; TGCAGGTCAGGGTTTTCCCAGTCAAGGCGCAT

5, 4; AGGCTGGCCCGGATATTCATTACCAGATTCAT

5, 5; CAGTTGAGACATAACGCCAAAAGGAAAATCAG

5, 6; GTCTTTACGCATCAAAAAGATTAAGAGGAAGC

5, 7; TACAGGCACCTCAGAGCATAAAGCTAGCTATT

5, 8; TTTGAGAGGTAGCGCGTTTTCATCCTTGAGCC

5, 9; ATTTGGGATTAACTGAACACCCTGTAATTTGC

6, 1; ATTTACATATTGCGTTGCGCTCACAAAAGAGT

6, 2; AGCAGAAGCGGCCAGTGCCAAGCTGAGCTAAC

6, 3; CGTAACAAGAACGGTGTACAGACCCGACGTTG

6, 4; GGCATAGTATTATTACAGGTAGAACAAATCAA

6, 5; CCGAAAGAGAATGACCATAAATCAAATTACGA

6, 6; TTGTACCAGTAGCATTAACATCCAATAAATCA

6, 7; CGGTCATATTAATGCCGGAGAGGGTAAATCGG

6, 8; CAGAGGGTTTATCACCGTCACCGAGGCATTTT

6, 9; GCTAACGAGCGTCTTTCCAGAGCCAACAAAGT




4

1, 5; CTCCAGCCTTTTAGGTTACGTTGGTGTATTGACCGT

1, 6; AATGGGATTTTTCTTCCTGTAGCCAGCTATAATTCG

1, 7; CGTCTGGCTTTTAACGTTAATATTTTGTAAATATTT

1, 8; AAATTGTATTTTAATCGTAAAACTAGCAAGAATCGA

1, 9; TGAACGGTTTTTAAACCATCGATAGCAGGGAAACGT

1, 10; CACCAATGTTTTATGAAAATAGCAGCCTTTGTTTAACGTCAAAA

2, 1; CCACACAATCGGCCAACGCGCGGGTTTAGACA

2, 2; AAGGGGGAATTCGTAATCATGGTCTCACAATT

2, 3; CCGGAAACCAACTGTTGGGAAGGGGCTGGCGA

2, 4; CATCGTAACGTATCGGCCTCAGGATTCTGGTG

2, 5; CATTAAATGGGAACAAACGGCGGAGATGGGCG

2, 6; GCGTTAAAAGGAACGCCATCAAAATTCATCAA

2, 7; ATATGTACGGAAGATTGTATAAGCTAAAATTC

2, 8; TCAGTAGCGTCTGGAGCAAACAAGTGTCAATC

2, 9; AGAATAACTACCATTAGCAAGGCCCACCGTAA

2, 10; CCAATCCAAATAAGAAACGATTTTTTACAGAG

3, 1; GGAACGGTACGCCAGAATCTTGAGCAGCTGCA

3, 2; TTAATGAACATACGAGCCGGAAGCCGGGTACC

3, 3; GAGCTCGATGTGCTGCAAGGCGATTAAGTTGG

3, 4; AGGCTGCGCAGGCAAAGCGCCATTTAATCTTC

3, 5; ACGACGACCCGTGCATCTGCCAGTTTCAACTA

3, 6; TTCTCCGTGTGAGCGAGTAACAACAGAAGCAA

3, 7; TCATTTTTTAACCAATTTTTTGTTAAATCAGC

3, 8; CAAAAACACCCGGTTGATAATCAGAAATTAAG

3, 9; GCCTGAGAGACAGAATCAAGTTTGTCACCAGT

3, 10; AGCACCATATAAAAACAGGGAAGCTATTTATC

5

4, 1; TAAAGCCTGGGAAACCTGTCGTGCAAGTGTTT

4, 2; GTAACGCCGACTCTAGAGGATCCCATAAAGTG

4, 3; GACAAGAATGACCTTCATCAAGAGCGCCATTC

4, 4; ATGCAGATATTTAGGAATACCACATTGAGGGG

4, 5; AGCGGATTCCTGACTATTATAGTCCCGTCGGA

4, 6; CAATAAAGAGGCAAAGAATTAGCAAAAAGCCC

4, 7; GTCAGACTATCTACAAAGGCTATCAGGTCATT

4, 8; CGGGAGAAATTAGAGCCAGCAAAACCTTTAGC

4, 9; CAGTTACAAAATAAACAGCCATATGCATTAGA

5, 1; TTATAATCAGTGAGGCCACCGAGTTGCCCGCT

5, 2; TTCCAGTCGGGGTGCCTAATGAGTTGCATGCC

5, 3; TGCAGGTCAGGGTTTTCCCAGTCAAGGCGCAT

5, 4; AGGCTGGCCCGGATATTCATTACCAGATTCAT

5, 5; CAGTTGAGACATAACGCCAAAAGGAAAATCAG

5, 6; GTCTTTACGCATCAAAAAGATTAAGAGGAAGC

5, 7; TACAGGCACCTCAGAGCATAAAGCTAGCTATT

5, 8; TTTGAGAGGTAGCGCGTTTTCATCCTTGAGCC

5, 9; ATTTGGGATTAACTGAACACCCTGTAATTTGC

6, 1; ATTTACATATTGCGTTGCGCTCACAAAAGAGT

6, 2; AGCAGAAGCGGCCAGTGCCAAGCTGAGCTAAC

6, 3; CGTAACAAGAACGGTGTACAGACCCGACGTTG

6, 4; GGCATAGTATTATTACAGGTAGAACAAATCAA

6, 5; CCGAAAGAGAATGACCATAAATCAAATTACGA

6, 6; TTGTACCAGTAGCATTAACATCCAATAAATCA

6, 7; CGGTCATATTAATGCCGGAGAGGGTAAATCGG

6, 8; CAGAGGGTTTATCACCGTCACCGAGGCATTTT

6, 9; GCTAACGAGCGTCTTTCCAGAGCCAACAAAGT




6

7, 1; CTGTCCATCACGCAAATTAACCGTAATGGATT

7, 2; TCACATTATGGCAGATTCACCAGTGAACCACC

7, 3; TAAAACGAATAAAACAGAGGTGAGTTGAAAGA

7, 4; GGACAGATAGCTGCTCATTCAGTGGAACTAAC

7, 5; GGAACAACAAGAGCAACACTATCAACAGTTCA

7, 6; GAAAACGACTTCAAATATCGCGTTTTAATTCG

7, 7; TAATAGTAAAAACATTATGACCCTCGTTCTAG

7, 8; CTGATAAAGCCCCCTTATTAGCGTATTCATTA

7, 9; AAGGTGAAAATTGAGCGCTAATATTTACCAAC

8, 1; CAGTAATAACGCTCAATCGTCTGATGTAGCAA

8, 2; TATTAACAATTAAAAATACCGAACCACACGAC

8, 3; TTGCCCTGACCGAACTGACCAACTGCGGTCAG

8, 4; GTTTACCATCTACGTTAATAAAACAATAAGGC

8, 5; AGCTTCAACCCTCAAATGCTTTAATAACCCTC

8, 6; CAACTAAAGTACGGTGACATGTTTTAAATATG

8, 7; TTTGCGGGCTGAAAAGGTGGCATCAATTCTAC

8, 8; TTTTCATACAATATGATATTCAACGTAATACT

8, 9; TAACCCACAATATTGACGGAAATTTTGCCATC

8, 10; CCAGCTACAATTTTATCCTGAATCCAGAGAGA

9, 1; TACTTCTTTGATTAGTAATAACATAAATACCT

9, 2; ACATTTTGAAAGGGACATTCTGGCGCCCTAAA

9, 3; ACATCGCCCCGCCTGCAACAGTGCGGTCAATC

9, 4; ATAAGGGAACGAGAACACCAGAACACGTTGGG

9, 5; AAGAAAAAGACGACGATAAAAACCAATATTCA

9, 6; TTGAATCCAGCGAACCAGACCGGACTGGTGCT

9, 7; GTAGCTCATCTGGAAGTTTCATTCTCATTTGG

7

9, 8; GGCGCGAGAGAAGCCTTTATTTCAACAGTCAA

9, 9; ATCACCATATCAAAATCACCGGAATGAGGGAG

9, 10; GGAAGGTAAAGAATTGAGTTAAGCTTTTGCAC

10, 1; ATAGAACCGGAAAAACGCTCATGGCACTTGCC

10, 2; GAGCCAGCAATGCGCGAACTGATACAACAGAG

10, 3; AATTGGGCGGAACGAGGCGCAGACCACGCTGA

10, 4; GAGAGGCTCATTATACCAGTCAGGGAGTAGTA

10, 5; CCAACAGGCTGCGGAATCGTCATAAAAATAGC

10, 6; AGTTGATTAGCTTAATTGCTGAATAGCAAACT

10, 7; ATAAAAATCTGTTTAGCTATATTTCATGTAAC

10, 8; ACCACCGGGTGAGAAAGGCCGGAGACGCAAGG

10, 9; AAGAGCAAGCGACATTCAACCGATCCAGAGCC

10, 10; GCCTTAAATCAAGATTAGTTGCTACCAATAAT

11, 1; TGAGTAGAAGAACTCAAACTATCGGCCAGCCA

11, 2; TTGCAACACTTCTGACCTGAAAGCATGGCTAT

11, 3; TAGTCTTTAGCAAATGAAAAATCTTCCATGTT

11, 4; ACTTAGCCTTGAGATGGTTTAATTATTTTAAG

11, 5; AACTGGCTTTTGCAAAAGAAGTTTTGGATAGC

11, 6; GTCCAATATCAGGATTAGAGAGTATTTGCGGA

11, 7; TGGCTTAGCCCAATTCTGCGAACGGCAAATGG

11, 8; TCAATAACTTTTAGAACCCTCATAGTAAAGAT

11, 9; TCAAAAGGAACCGCCTCCCTCAGACGCCAAAG

11, 10; ACAAAAGGGAAACAATGAAATAGCGTTTTGAA

12, 1; ACGTGGCATTTTCCAGAACAATATTACCGCCTTGCTGGTAATAT

12, 2; ACCTTGCTCAGACAATATTTTTGAGTAAGAAT

12, 3; ATTGTGTCGAAATCCGCGACCTGCAAAGCATC




6

7, 1; CTGTCCATCACGCAAATTAACCGTAATGGATT

7, 2; TCACATTATGGCAGATTCACCAGTGAACCACC

7, 3; TAAAACGAATAAAACAGAGGTGAGTTGAAAGA

7, 4; GGACAGATAGCTGCTCATTCAGTGGAACTAAC

7, 5; GGAACAACAAGAGCAACACTATCAACAGTTCA

7, 6; GAAAACGACTTCAAATATCGCGTTTTAATTCG

7, 7; TAATAGTAAAAACATTATGACCCTCGTTCTAG

7, 8; CTGATAAAGCCCCCTTATTAGCGTATTCATTA

7, 9; AAGGTGAAAATTGAGCGCTAATATTTACCAAC

8, 1; CAGTAATAACGCTCAATCGTCTGATGTAGCAA

8, 2; TATTAACAATTAAAAATACCGAACCACACGAC

8, 3; TTGCCCTGACCGAACTGACCAACTGCGGTCAG

8, 4; GTTTACCATCTACGTTAATAAAACAATAAGGC

8, 5; AGCTTCAACCCTCAAATGCTTTAATAACCCTC

8, 6; CAACTAAAGTACGGTGACATGTTTTAAATATG

8, 7; TTTGCGGGCTGAAAAGGTGGCATCAATTCTAC

8, 8; TTTTCATACAATATGATATTCAACGTAATACT

8, 9; TAACCCACAATATTGACGGAAATTTTGCCATC

8, 10; CCAGCTACAATTTTATCCTGAATCCAGAGAGA

9, 1; TACTTCTTTGATTAGTAATAACATAAATACCT

9, 2; ACATTTTGAAAGGGACATTCTGGCGCCCTAAA

9, 3; ACATCGCCCCGCCTGCAACAGTGCGGTCAATC

9, 4; ATAAGGGAACGAGAACACCAGAACACGTTGGG

9, 5; AAGAAAAAGACGACGATAAAAACCAATATTCA

9, 6; TTGAATCCAGCGAACCAGACCGGACTGGTGCT

9, 7; GTAGCTCATCTGGAAGTTTCATTCTCATTTGG

7

9, 8; GGCGCGAGAGAAGCCTTTATTTCAACAGTCAA

9, 9; ATCACCATATCAAAATCACCGGAATGAGGGAG

9, 10; GGAAGGTAAAGAATTGAGTTAAGCTTTTGCAC

10, 1; ATAGAACCGGAAAAACGCTCATGGCACTTGCC

10, 2; GAGCCAGCAATGCGCGAACTGATACAACAGAG

10, 3; AATTGGGCGGAACGAGGCGCAGACCACGCTGA

10, 4; GAGAGGCTCATTATACCAGTCAGGGAGTAGTA

10, 5; CCAACAGGCTGCGGAATCGTCATAAAAATAGC

10, 6; AGTTGATTAGCTTAATTGCTGAATAGCAAACT

10, 7; ATAAAAATCTGTTTAGCTATATTTCATGTAAC

10, 8; ACCACCGGGTGAGAAAGGCCGGAGACGCAAGG

10, 9; AAGAGCAAGCGACATTCAACCGATCCAGAGCC

10, 10; GCCTTAAATCAAGATTAGTTGCTACCAATAAT

11, 1; TGAGTAGAAGAACTCAAACTATCGGCCAGCCA

11, 2; TTGCAACACTTCTGACCTGAAAGCATGGCTAT

11, 3; TAGTCTTTAGCAAATGAAAAATCTTCCATGTT

11, 4; ACTTAGCCTTGAGATGGTTTAATTATTTTAAG

11, 5; AACTGGCTTTTGCAAAAGAAGTTTTGGATAGC

11, 6; GTCCAATATCAGGATTAGAGAGTATTTGCGGA

11, 7; TGGCTTAGCCCAATTCTGCGAACGGCAAATGG

11, 8; TCAATAACTTTTAGAACCCTCATAGTAAAGAT

11, 9; TCAAAAGGAACCGCCTCCCTCAGACGCCAAAG

11, 10; ACAAAAGGGAAACAATGAAATAGCGTTTTGAA

12, 1; ACGTGGCATTTTCCAGAACAATATTACCGCCTTGCTGGTAATAT

12, 2; ACCTTGCTCAGACAATATTTTTGAGTAAGAAT

12, 3; ATTGTGTCGAAATCCGCGACCTGCAAAGCATC




8

12, 4; CAGCATCGTGAATTACCTTATGCGTCAACTTT

12, 5; TTTTCACGAGTAAAATGTTTAGACTGCCAGAG

12, 6; AGGAACCCTTGATAAGAGGTCATTCCTTTAAT

12, 7; CTCAGGAGCCATTAGATACATTTCAGTAGATT

12, 8; TAATAAGTCCTGAGTAATGTGTAGTATTTTAA

12, 9; TCTTACCGTCATATGGTTTACCAGGCCGCCAC

12, 10; AGCGAACCTCCCGACTTGCGGGAGAATAGCTA

13, 1; GAACCTCAAATATCAACCTGATAA

13, 2; AATCATTGGAACGAGGGTAGCAACGCGAAAGA

13, 3; GGGGTAATTTGAAAATCTCCAAAATAATAATT

13, 4; TGCTCCTTATGTACCGTAACACTGAGCCCAAT

13, 5; TAGTTTGAGTTTAGTACCGCCACCTCACCGTA

13, 6; ATGCAATGTTTAACGGGGTCAGTGTGTACTGG

13, 7; CCTCAGAACCGCCACCCTCAGAGCACAATCAA

13, 8; TAGAAAATAAGCCCTTTTTAAGAAGGCGTTTT

14, 1; AGGCTTTGGAGATTTGTATCATCGACCCTCAA

14, 2; CCAAAAGGATCGTCACCCTCAGCAGGCTACAG

14, 3; CACCAGTACTAAAGGAATTGCGAAAAAAGGCT

14, 4; CGCCACCCATTTTCAGGGATAGCAAGTTTCGT

14, 5; AACAGTGCGCCCGGAATAGGTGTACTCAGAAC

14, 6; TCAGAGCCTTTGATGATACAGGAGCCTTGAGT

14, 7; AGATAGCCATAAGTTTATTTTGTCCACCACCC

14, 8; TTATCCGGTATTCTAAGAACGCGAAAGTAAGC

15, 1; TCAATATCTGGTCAGTTGGCAAATGAAACAAA

15, 2; GTACAACGAGGACTAAAGACTTTTAGGCCGCT

15, 3; TTTGCGGGAGCCTTTAATTGTATCGAATAGAA

9

15, 4; AGGAACAACAAACTACAACGCCTGTAGCATTC

15, 5; CAGAGCCACCACCCTCTCAGAACCGCCACCCT

15, 6; TAAGTATACCGTATAAACAGTTAAAGCGTCAT

15, 7; ACATGGCTGCCACCAGAACCACCAGCAAAGAC

15, 8; ACCACGGAGAACAAAGTTACCAGATAGAAGGC

16, 1; AAGTTTCCCGATTATACCAAGCGCCAACAGTA

16, 2; AGCTTGCTGCTTGCAGGGAGTTAATCATGAGG

16, 3; CACAGACAAGTTTCAGCGGAGTGAGGTTTATC

16, 4; GCCTATTTTAAGTGCCGTCGAGAGGGTTGATA

16, 5; GCCGCCAGATTTACCGTTCCAGTATGCCCCCT

16, 6; GAGGAAACAACATATAAAAGAAACCCAGAGCC

16, 7; GCCCAATAGCAAGCAAATCAGATAAGGAAACC

17, 1; GAAAGGAATTGAGGAAGGTTATCTCATCTTTG

17, 2; ACCCCCAGATTAAACGGGTAAAATATATTCGG

17, 3; TCGCTGAGTTCGAGGTGAATTTCTCTAAACAA

17, 4; CTTTCAACGCCCTCATAGTTAGCGTAACGATC

17, 5; CAGGCGGACGGAACCTATTATTCTAAAGCGCA

17, 6; GTCTCTGACATTGACAGGAGGTTGCATACATA

17, 7; AAGGTGGCGCAATAATAACGGAATATTACCGC

18, 1; CCACTACGAATACACTAAAACACTAAAATATC

18, 2; TTGATACCCCCACGCATAACCGATACGTAATG

18, 3; TAAAGTTTTCTGTATGGGATTTTGTAAACAGC

18, 4; AAAGTATTGATTAGCGGGGTTTTGCTCAGTAC

18, 5; CAGACGATATTAAAGCCAGAATGGGAAACATG

18, 6; GAACTGGCGCAAACGTAGAAAATAAGGCAGGT

18, 7; GTTTTTATTTTCATCGTAGGAATCACCCAAAA




8

12, 4; CAGCATCGTGAATTACCTTATGCGTCAACTTT

12, 5; TTTTCACGAGTAAAATGTTTAGACTGCCAGAG

12, 6; AGGAACCCTTGATAAGAGGTCATTCCTTTAAT

12, 7; CTCAGGAGCCATTAGATACATTTCAGTAGATT

12, 8; TAATAAGTCCTGAGTAATGTGTAGTATTTTAA

12, 9; TCTTACCGTCATATGGTTTACCAGGCCGCCAC

12, 10; AGCGAACCTCCCGACTTGCGGGAGAATAGCTA

13, 1; GAACCTCAAATATCAACCTGATAA

13, 2; AATCATTGGAACGAGGGTAGCAACGCGAAAGA

13, 3; GGGGTAATTTGAAAATCTCCAAAATAATAATT

13, 4; TGCTCCTTATGTACCGTAACACTGAGCCCAAT

13, 5; TAGTTTGAGTTTAGTACCGCCACCTCACCGTA

13, 6; ATGCAATGTTTAACGGGGTCAGTGTGTACTGG

13, 7; CCTCAGAACCGCCACCCTCAGAGCACAATCAA

13, 8; TAGAAAATAAGCCCTTTTTAAGAAGGCGTTTT

14, 1; AGGCTTTGGAGATTTGTATCATCGACCCTCAA

14, 2; CCAAAAGGATCGTCACCCTCAGCAGGCTACAG

14, 3; CACCAGTACTAAAGGAATTGCGAAAAAAGGCT

14, 4; CGCCACCCATTTTCAGGGATAGCAAGTTTCGT

14, 5; AACAGTGCGCCCGGAATAGGTGTACTCAGAAC

14, 6; TCAGAGCCTTTGATGATACAGGAGCCTTGAGT

14, 7; AGATAGCCATAAGTTTATTTTGTCCACCACCC

14, 8; TTATCCGGTATTCTAAGAACGCGAAAGTAAGC

15, 1; TCAATATCTGGTCAGTTGGCAAATGAAACAAA

15, 2; GTACAACGAGGACTAAAGACTTTTAGGCCGCT

15, 3; TTTGCGGGAGCCTTTAATTGTATCGAATAGAA

9

15, 4; AGGAACAACAAACTACAACGCCTGTAGCATTC

15, 5; CAGAGCCACCACCCTCTCAGAACCGCCACCCT

15, 6; TAAGTATACCGTATAAACAGTTAAAGCGTCAT

15, 7; ACATGGCTGCCACCAGAACCACCAGCAAAGAC

15, 8; ACCACGGAGAACAAAGTTACCAGATAGAAGGC

16, 1; AAGTTTCCCGATTATACCAAGCGCCAACAGTA

16, 2; AGCTTGCTGCTTGCAGGGAGTTAATCATGAGG

16, 3; CACAGACAAGTTTCAGCGGAGTGAGGTTTATC

16, 4; GCCTATTTTAAGTGCCGTCGAGAGGGTTGATA

16, 5; GCCGCCAGATTTACCGTTCCAGTATGCCCCCT

16, 6; GAGGAAACAACATATAAAAGAAACCCAGAGCC

16, 7; GCCCAATAGCAAGCAAATCAGATAAGGAAACC

17, 1; GAAAGGAATTGAGGAAGGTTATCTCATCTTTG

17, 2; ACCCCCAGATTAAACGGGTAAAATATATTCGG

17, 3; TCGCTGAGTTCGAGGTGAATTTCTCTAAACAA

17, 4; CTTTCAACGCCCTCATAGTTAGCGTAACGATC

17, 5; CAGGCGGACGGAACCTATTATTCTAAAGCGCA

17, 6; GTCTCTGACATTGACAGGAGGTTGCATACATA

17, 7; AAGGTGGCGCAATAATAACGGAATATTACCGC

18, 1; CCACTACGAATACACTAAAACACTAAAATATC

18, 2; TTGATACCCCCACGCATAACCGATACGTAATG

18, 3; TAAAGTTTTCTGTATGGGATTTTGTAAACAGC

18, 4; AAAGTATTGATTAGCGGGGTTTTGCTCAGTAC

18, 5; CAGACGATATTAAAGCCAGAATGGGAAACATG

18, 6; GAACTGGCGCAAACGTAGAAAATAAGGCAGGT

18, 7; GTTTTTATTTTCATCGTAGGAATCACCCAAAA




10

19, 1; TTTAGGTGCACTAACAACTAATAGATTAGAGC

19, 2; GGCGAAAGAAGGCACCAACCTAAAGAATTATC

19, 3; AACCATCGGATAGTTGCGCCGACATAACGTCA

19, 4; ATGAATTTTGTCGTCTTTCCAGACAACAAAAT

19, 5; AGGATTAGAAGAGGCTGAGACTCCTTAGTTAA

19, 6; GGATCTTCTGGCCTTGATATTCACGCTCAACA

19, 7; GTATGTTAATGATTAAGACTCCTTTGCAGAGC

20, 1; ATCATATTACCACCAGAAGGAGCGACGAAAGA

20, 2; GATGAATACGTAGATTTTCAGGTTATGACAAC

20, 3; TAATTACAAAACAAACATCAAGAAGTTAGTAA

20, 4; AAGACGCTGAGAAGAGAGCGATAGCTTAGATT

20, 5; TTTCATCTCTTTTTCAAATATATTTCAAGAGA

20, 6; GTAGGGCTCCAGTATAAAGCCAACAAACGAAT

20, 7; GCGCCTGTCAACATGTTCAGCTAAATTACGCA

20, 8; CAAGTACCGCACTCATCGAGAACAAGCAAGCC

21, 1; CGTCAATAGATAATACATTTGAGGTTTGCGGA

21, 2; ACAAAGAACCTGATTATCAGATGAAGAAATAA

21, 3; AGAAATTGTACAGTAACAGTACCTGCAAAAGA

21, 4; AGATGATGTTTAACAATTTCATTTGAATCCTT

21, 5; GAAAACATTCAATAGTGAATTTATAAAGAACG

21, 6; CGAGAAAATCTGACCTAAATTTAAGTTATACA

21, 7; AATTCTTATAATTGAGAATCGCCAAGACGACG

21, 8; ACAATAAATTATCAACAATAGATATATTAAAC

22, 1; CATCAATATGAGTAACATTATCATATTTAGAA

22, 2; GGGAGAAATATTAGCACGTAAAACTGGCAATT

11

22, 3; TTTTTTAACATTTCAATTACCTGATTTACATC

22, 4; TAGGTCTGAATTAATTTTCCCTTAGAATTACC

22, 5; AATACCGAAATCCAATCGCAAGACCAAAATCA

22, 6; AACGCCAAGTTTAGTATCATATGCTGGTTTGA

22, 7; ACAAGAAATAAAGTAATTCTGTCCTATTTAAC

22, 8; TTTCCTTATCATTCCAAGAACGGGAGTCCTGA

23, 1; GTATTAGACTTTACAAACAATTCGATTAATTT

23, 2; TAAAAGTTTAATCCTGATTGTTTGCCAACCAT

23, 3; ATCAAAATCAATAACGGATTCGCCGCAGAGGC

23, 4; GAATTATTTGGAAACAGTACATAAGTAAATCG

23, 5; TCGCTATTAGAGACTACCTTTTTATGTAAATG

23, 6; CTGATGCACCGTGTGATAAATAAGTTACTAGA

23, 7; AAAAGCCTCATGTAATTTAGGCAGAAGTACCG

23, 8; ACAAAAGGAATAATATCCCATCCTCGGCTGTC

24, 1; TTCTGAATTTTTCCTTTGCCCGAACGTTACAACTCGTATTAAAT

24, 2; TTGAATACTTTTTATGGAAGGAATTGAAGATTATAC

24, 3; ATGTGAGTTTTTCAAGTTACAAAATCGCTGATTGCT

24, 4; CTTAGGTTTTTTGAATAACCTTGCTTCTATCAATAT

24, 5; TAAGAATATTTTGGGTTATATAACTATAACCTCCGG

24, 6; TCGAGCCATTTTAACACCGGAATCATAAGCGTTAAA

24, 7; AGCATGTATTTTGTAATAAGAGAATATAAGGCATTT

24, 8; GAAACCAATCAATAATAATTTACG




10

19, 1; TTTAGGTGCACTAACAACTAATAGATTAGAGC

19, 2; GGCGAAAGAAGGCACCAACCTAAAGAATTATC

19, 3; AACCATCGGATAGTTGCGCCGACATAACGTCA

19, 4; ATGAATTTTGTCGTCTTTCCAGACAACAAAAT

19, 5; AGGATTAGAAGAGGCTGAGACTCCTTAGTTAA

19, 6; GGATCTTCTGGCCTTGATATTCACGCTCAACA

19, 7; GTATGTTAATGATTAAGACTCCTTTGCAGAGC

20, 1; ATCATATTACCACCAGAAGGAGCGACGAAAGA

20, 2; GATGAATACGTAGATTTTCAGGTTATGACAAC

20, 3; TAATTACAAAACAAACATCAAGAAGTTAGTAA

20, 4; AAGACGCTGAGAAGAGAGCGATAGCTTAGATT

20, 5; TTTCATCTCTTTTTCAAATATATTTCAAGAGA

20, 6; GTAGGGCTCCAGTATAAAGCCAACAAACGAAT

20, 7; GCGCCTGTCAACATGTTCAGCTAAATTACGCA

20, 8; CAAGTACCGCACTCATCGAGAACAAGCAAGCC

21, 1; CGTCAATAGATAATACATTTGAGGTTTGCGGA

21, 2; ACAAAGAACCTGATTATCAGATGAAGAAATAA

21, 3; AGAAATTGTACAGTAACAGTACCTGCAAAAGA

21, 4; AGATGATGTTTAACAATTTCATTTGAATCCTT

21, 5; GAAAACATTCAATAGTGAATTTATAAAGAACG

21, 6; CGAGAAAATCTGACCTAAATTTAAGTTATACA

21, 7; AATTCTTATAATTGAGAATCGCCAAGACGACG

21, 8; ACAATAAATTATCAACAATAGATATATTAAAC

22, 1; CATCAATATGAGTAACATTATCATATTTAGAA

22, 2; GGGAGAAATATTAGCACGTAAAACTGGCAATT

11

22, 3; TTTTTTAACATTTCAATTACCTGATTTACATC

22, 4; TAGGTCTGAATTAATTTTCCCTTAGAATTACC

22, 5; AATACCGAAATCCAATCGCAAGACCAAAATCA

22, 6; AACGCCAAGTTTAGTATCATATGCTGGTTTGA

22, 7; ACAAGAAATAAAGTAATTCTGTCCTATTTAAC

22, 8; TTTCCTTATCATTCCAAGAACGGGAGTCCTGA

23, 1; GTATTAGACTTTACAAACAATTCGATTAATTT

23, 2; TAAAAGTTTAATCCTGATTGTTTGCCAACCAT

23, 3; ATCAAAATCAATAACGGATTCGCCGCAGAGGC

23, 4; GAATTATTTGGAAACAGTACATAAGTAAATCG

23, 5; TCGCTATTAGAGACTACCTTTTTATGTAAATG

23, 6; CTGATGCACCGTGTGATAAATAAGTTACTAGA

23, 7; AAAAGCCTCATGTAATTTAGGCAGAAGTACCG

23, 8; ACAAAAGGAATAATATCCCATCCTCGGCTGTC

24, 1; TTCTGAATTTTTCCTTTGCCCGAACGTTACAACTCGTATTAAAT

24, 2; TTGAATACTTTTTATGGAAGGAATTGAAGATTATAC

24, 3; ATGTGAGTTTTTCAAGTTACAAAATCGCTGATTGCT

24, 4; CTTAGGTTTTTTGAATAACCTTGCTTCTATCAATAT

24, 5; TAAGAATATTTTGGGTTATATAACTATAACCTCCGG

24, 6; TCGAGCCATTTTAACACCGGAATCATAAGCGTTAAA

24, 7; AGCATGTATTTTGTAATAAGAGAATATAAGGCATTT

24, 8; GAAACCAATCAATAATAATTTACG




12

Figures S1-S6. Molecular Details of the Molecules used in this Work.

Figure S1. The Strand Structure of the M13 Origami Used in This Work.

13

Figure S2. The Sequence of the Triangle Capture Molecule that Points Towards the Notch.




12

Figures S1-S6. Molecular Details of the Molecules used in this Work.

Figure S1. The Strand Structure of the M13 Origami Used in This Work.

13

Figure S2. The Sequence of the Triangle Capture Molecule that Points Towards the Notch.




14

Figure S3. The Sequence of the Triangle Capture Molecule that Points Away from the Notch.




14

Figure S3. The Sequence of the Triangle Capture Molecule that Points Away from the Notch.

15

Figure S4. The Sequence of the Diamond-Shaped Capture Molecule.




16

Figure S5. The Sequence of the Linear Capture Molecule.

18

Figure S6. The Sequences of the two Cassettes in the PX and JX2 States.

Cassette1 is shown on the top and Cassette 2 is on the bottom. The strands which set the cassette to the PX or JX2 state were labeled as Set-P1/2 or Set-J1/2, respectively. The strands that are completely complementary to the set strands were labeled as Fuel-P1/2 or Fuel-J1/2, respectively.




16

Figure S5. The Sequence of the Linear Capture Molecule.

18

Figure S6. The Sequences of the two Cassettes in the PX and JX2 States.

Cassette1 is shown on the top and Cassette 2 is on the bottom. The strands which set the cassette to the PX or JX2 state were labeled as Set-P1/2 or Set-J1/2, respectively. The strands that are completely complementary to the set strands were labeled as Fuel-P1/2 or Fuel-J1/2, respectively.




19

Figure S7. Non-Denaturing Gels of Components of the System.

All images are 5% non-denaturing gels. (a) was run at 25 ˚C, but (b) and (c) were run at 4 ˚C. Panel (a) illustrates the formation of the cassettes. Lane 1 contains a 100 nucleotide pair marker, lane 2 contains cassette 1 in the JX2 state, lane 3 contains cassette 1 in the PX state, lane 4 contains cassette 2 in the JX2 state and lane 5 contains cassette 2 in the PX state. Lanes 2-5 contain 20 µL of each cassette at a

concentration of 50 nM. Panel (b) contains the capture molecules. Lane 6 contains the triangle capture molecule that points towards the notch, lane 7 contains the triangle capture molecule that points away from the notch, lane 8 contains a 100 nucleotide pair marker, lane 9 contains the diamond capture molecule and lane 10 contains the linear capture molecule. Lanes 6, 7, 9 and 10 contain 20 µL of each

capture molecule at a concentration of 50 nM. Panel (c) demonstrates binding between the linear capture molecule and the cassettes. Lane 11 contains the linear capture molecule, lane 12 contains cassette 1 in the JX2 state, lane 13 contains cassette 2 in the JX2 state, lane 14 contains cassette 1 + cassette 2 + the linear capture molecule, lane 15 contains cassette 1 + the linear capture molecule, lane 16 contains cassette 2 + the linear capture molecule and lane 17 contains a 100 nucleotide pair marker. Lanes 11-13 contain 20 µL of each molecule at a concentration of 50 nM. The contents of lane 14, were

produced as follows: 20 µL each of cassette 1, cassette 2 and the linear capture molecule at 50 nM

concentration were mixed together in a tube at room temperature, then heated up to 40 ˚C. and slowly cooled down to room temperature in a 2 L water bath over 24 hours. 20 µL of the mixture solution was

then taken out of the tube and loaded into the well for running the gel. The contents of lane 15 were prepared by mixing 20 µL each of cassette 1 and the linear capture molecule at 50 nM concentration at

room temperature. The contents of lane 16 lane were prepared by mixing, 20 µL each of cassette 2 and

20

the linear capture molecule at 50 nM concentration at room temperature. The mixtures for lanes 15 and 16 were then treated by exactly the same protocol as in Lane 14.

Figures S8-S10. Error Correction of Three Components.




19

Figure S7. Non-Denaturing Gels of Components of the System.

All images are 5% non-denaturing gels. (a) was run at 25 ˚C, but (b) and (c) were run at 4 ˚C. Panel (a) illustrates the formation of the cassettes. Lane 1 contains a 100 nucleotide pair marker, lane 2 contains cassette 1 in the JX2 state, lane 3 contains cassette 1 in the PX state, lane 4 contains cassette 2 in the JX2 state and lane 5 contains cassette 2 in the PX state. Lanes 2-5 contain 20 µL of each cassette at a

concentration of 50 nM. Panel (b) contains the capture molecules. Lane 6 contains the triangle capture molecule that points towards the notch, lane 7 contains the triangle capture molecule that points away from the notch, lane 8 contains a 100 nucleotide pair marker, lane 9 contains the diamond capture molecule and lane 10 contains the linear capture molecule. Lanes 6, 7, 9 and 10 contain 20 µL of each

capture molecule at a concentration of 50 nM. Panel (c) demonstrates binding between the linear capture molecule and the cassettes. Lane 11 contains the linear capture molecule, lane 12 contains cassette 1 in the JX2 state, lane 13 contains cassette 2 in the JX2 state, lane 14 contains cassette 1 + cassette 2 + the linear capture molecule, lane 15 contains cassette 1 + the linear capture molecule, lane 16 contains cassette 2 + the linear capture molecule and lane 17 contains a 100 nucleotide pair marker. Lanes 11-13 contain 20 µL of each molecule at a concentration of 50 nM. The contents of lane 14, were

produced as follows: 20 µL each of cassette 1, cassette 2 and the linear capture molecule at 50 nM

concentration were mixed together in a tube at room temperature, then heated up to 40 ˚C. and slowly cooled down to room temperature in a 2 L water bath over 24 hours. 20 µL of the mixture solution was

then taken out of the tube and loaded into the well for running the gel. The contents of lane 15 were prepared by mixing 20 µL each of cassette 1 and the linear capture molecule at 50 nM concentration at

room temperature. The contents of lane 16 lane were prepared by mixing, 20 µL each of cassette 2 and

20

the linear capture molecule at 50 nM concentration at room temperature. The mixtures for lanes 15 and 16 were then treated by exactly the same protocol as in Lane 14.

Figures S8-S10. Error Correction of Three Components.




21

Figure S8. The Correction Procedure Applied to the Linear Capture Molecule

Panel (a) contains the origami to which the mixture has been applied. (b) contains the material in (a), but to which the diamond capture molecule has been applied. (c) contains the material in (b), but to which the linear capture molecule has been applied. (d) contains the material in (c), but to which the triangle pointing towards the notch has been applied, and (e) contains the material in (d), but to which the triangle pointing away from the notch has been applied. The thermal protocol has been applied between each step. Panels (f), (g), (h) and (i) change the order: The triangle away from the notch, the triangle towards the notch, the line and the diamond have applied, respectively in these panels. Panels (c), (d) and (e) contain exclusively linear elements. Similarly, panels (h) and (i) contain exclusively linear elements. Arrow conventions of Figure 3 apply.

22

Figure S9. The Correction Procedure Applied to the Triangle Capture Molecule Pointing Away

from the Notch

Panel (a) contains the origami to which the mixture has been applied. (b) contains the material in (a), but to which the diamond capture molecule has been applied. (c) contains the material in (b), but to which the triangle capture molecule pointing towards the notch has been applied. (d) contains the material in (c), but to which the linear capture molecule has been applied, and (e) contains the material in (d), but to which the triangle pointing away from the notch has been applied. The thermal protocol has been applied between each step. Panels (f), (g), (h) and (i) change the order: The linear capture molecule, the triangle away from the notch, the diamond and the triangle towards the notch have been applied, respectively in these panels. Panel (e) in the first series and panels (g), (h) and (i) in the second series contain exclusively triangles pointing away from the notch. Arrow conventions of Figure 3 apply.




21

Figure S8. The Correction Procedure Applied to the Linear Capture Molecule

Panel (a) contains the origami to which the mixture has been applied. (b) contains the material in (a), but to which the diamond capture molecule has been applied. (c) contains the material in (b), but to which the linear capture molecule has been applied. (d) contains the material in (c), but to which the triangle pointing towards the notch has been applied, and (e) contains the material in (d), but to which the triangle pointing away from the notch has been applied. The thermal protocol has been applied between each step. Panels (f), (g), (h) and (i) change the order: The triangle away from the notch, the triangle towards the notch, the line and the diamond have applied, respectively in these panels. Panels (c), (d) and (e) contain exclusively linear elements. Similarly, panels (h) and (i) contain exclusively linear elements. Arrow conventions of Figure 3 apply.

22

Figure S9. The Correction Procedure Applied to the Triangle Capture Molecule Pointing Away

from the Notch

Panel (a) contains the origami to which the mixture has been applied. (b) contains the material in (a), but to which the diamond capture molecule has been applied. (c) contains the material in (b), but to which the triangle capture molecule pointing towards the notch has been applied. (d) contains the material in (c), but to which the linear capture molecule has been applied, and (e) contains the material in (d), but to which the triangle pointing away from the notch has been applied. The thermal protocol has been applied between each step. Panels (f), (g), (h) and (i) change the order: The linear capture molecule, the triangle away from the notch, the diamond and the triangle towards the notch have been applied, respectively in these panels. Panel (e) in the first series and panels (g), (h) and (i) in the second series contain exclusively triangles pointing away from the notch. Arrow conventions of Figure 3 apply.




23

Figure S10. The Correction Procedure Applied to the Triangle Capture Molecule Pointing

Towards the Notch

Panel (a) contains the origami to which the mixture has been applied. (b) contains the material in (a), but to which the triangle away from the notch has been applied. (c) contains the material in (b), but to which the triangle capture molecule pointing towards the notch has been applied. (d) contains the material in (c), but to which the linear capture element has been applied, and (e) contains the material in (d), but to which the diamond capture molecule has been applied. The thermal protocol has been applied between each step. Panels (f), (g), (h) and (i) change the order: The linear capture molecule, the diamond, the triangle towards the notch and the triangle away from the notch have been applied, respectively in these panels. In the first series, panels (c), (d) and (e) contain exclusively triangles pointing towards the notch; in the second series, panels (h) and (i) contain only triangles pointing towards the notch. Arrow conventions of Figure 3 apply.

24

Figure S11. The Correction Procedure Applied to the Linear Feature, but Without an Initial

Four-Way Competition

Panel (a) contains the origami to which the triangle pointing away from the notch (half-right) has been applied. (b) contains the material in (a), but to which the triangle towards the notch has been applied. (c) contains the material in (b), but to which the linear capture feature has been applied. (d) contains the material in (c), but to which the diamond capture element has been applied. As soon as the correct element is applied, all the correct species are present. Arrow conventions of Figure 3 apply.




23

Figure S10. The Correction Procedure Applied to the Triangle Capture Molecule Pointing

Towards the Notch

Panel (a) contains the origami to which the mixture has been applied. (b) contains the material in (a), but to which the triangle away from the notch has been applied. (c) contains the material in (b), but to which the triangle capture molecule pointing towards the notch has been applied. (d) contains the material in (c), but to which the linear capture element has been applied, and (e) contains the material in (d), but to which the diamond capture molecule has been applied. The thermal protocol has been applied between each step. Panels (f), (g), (h) and (i) change the order: The linear capture molecule, the diamond, the triangle towards the notch and the triangle away from the notch have been applied, respectively in these panels. In the first series, panels (c), (d) and (e) contain exclusively triangles pointing towards the notch; in the second series, panels (h) and (i) contain only triangles pointing towards the notch. Arrow conventions of Figure 3 apply.

24

Figure S11. The Correction Procedure Applied to the Linear Feature, but Without an Initial

Four-Way Competition

Panel (a) contains the origami to which the triangle pointing away from the notch (half-right) has been applied. (b) contains the material in (a), but to which the triangle towards the notch has been applied. (c) contains the material in (b), but to which the linear capture feature has been applied. (d) contains the material in (c), but to which the diamond capture element has been applied. As soon as the correct element is applied, all the correct species are present. Arrow conventions of Figure 3 apply.




26

Table S1. Quantitation of the Correction Procedure

Results from 10 Images in Each Case

Triangle-F = Triangle Pointing Away from Notch

Triangle-T = Triangle Pointing towards Notch

Diamond (Figure 3) JX2-PX

Figure Panel Diamond Line Triangle-F Triangle-T Damaged Total

a 10 23 0 22 13 68

b 7 46 0 7 11 71

c 6 28 0 36 14 84

d 31 0 0 0 21 52

e 26 0 0 0 23 49

f 8 18 0 29 13 68

g 7 19 0 27 15 68

h 7 35 0 15 13 70

i 33 0 0 0 18 51

27

Line (Figure S8) JX2-JX2


a 8 32 19 0 23 82

b 19 13 5 0 26 63

c 0 65 0 0 18 83

d 0 57 0 0 21 78

e 0 43 0 0 17 60

f 4 26 23 0 28 81

g 2 17 21 0 16 56

h 0 48 0 0 13 61

i 0 53 0 0 13 66

Triangle-F (Figure S9) PX-JX2


a 0 16 18 24 19 77

b 0 8 21 16 24 69

c 0 6 13 28 23 70

d 0 15 11 23 18 67

e 0 0 37 0 22 59

f 0 38 11 6 24 79

g 0 0 44 0 28 72

h 0 0 42 0 23 65

i 0 0 28 0 19 47




26

Table S1. Quantitation of the Correction Procedure

Results from 10 Images in Each Case

Triangle-F = Triangle Pointing Away from Notch

Triangle-T = Triangle Pointing towards Notch

Diamond (Figure 3) JX2-PX


a 10 23 0 22 13 68

b 7 46 0 7 11 71

c 6 28 0 36 14 84

d 31 0 0 0 21 52

e 26 0 0 0 23 49

f 8 18 0 29 13 68

g 7 19 0 27 15 68

h 7 35 0 15 13 70

i 33 0 0 0 18 51

27

Line (Figure S8) JX2-JX2


a 8 32 19 0 23 82

b 19 13 5 0 26 63

c 0 65 0 0 18 83

d 0 57 0 0 21 78

e 0 43 0 0 17 60

f 4 26 23 0 28 81

g 2 17 21 0 16 56

h 0 48 0 0 13 61

i 0 53 0 0 13 66

Triangle-F (Figure S9) PX-JX2


a 0 16 18 24 19 77

b 0 8 21 16 24 69

c 0 6 13 28 23 70

d 0 15 11 23 18 67

e 0 0 37 0 22 59

f 0 38 11 6 24 79

g 0 0 44 0 28 72

h 0 0 42 0 23 65

i 0 0 28 0 19 47




28

Triangle-T (Figure S10) PX-PX


a 12 0 14 27 21 74

b 6 0 29 23 26 84

c 0 0 0 31 17 48

d 0 0 0 39 22 61

e 0 0 0 35 21 56

f 7 0 17 25 14 63

g 6 0 13 19 14 52

h 0 0 0 44 29 73

i 0 0 0 37 21 58

Those rows expected to be free of all incorrect components are shown in bold.

References

1. Seeman, N.C., De Novo Design of sequences for nucleic acid structure engineering. J. Biomol.

Str. & Dyns. 8, 573-581 (1990).

2. Ke, Y., Lindsay, S., Chang, Y, Liu, Y, Yan, H., Self-assembled water-soluble nucleic acid probe

molecules for label-free RNA hybridization. Science 319, 180-183 (2008).


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SUPPLEMENTARY INFORMATION...origami tiles was mixed with 4 µL each of a 50 nm solutions containing cassettes 1 and 2. The system ... then the solution was left at 25 ˚C for 2 hours