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S1 Synergy of Two Assembly Languages in DNA Nanostructures: Self-Assembly of Sequence-Defined Polymers on DNA Cages Pongphak Chidchob, Thomas G. W. Edwardson, Christopher J. Serpell, & Hanadi F. Sleiman* Department of Chemistry and Centre for Self-Assembled Chemical Structures (CSACS-CRMAA), McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada Supplementary Information Contents I. General S2 II. Instrumentation S2 III. Solid-phase synthesis and purification S3 IV. DNA sequences and characterization S4 V. Cage design and assembly S13 VI. Cage assembly with HEn-DNA S15 VII. Determination of cage aggregation numbers S20 VIII. Gold nanoparticle labeling on C4/HE6-DNA S23 IX. Cage assembly with HE/HEG-DNA S28 X. Assembly characterization on agarose gel electrophoresis S33 XI. Particle size distributions by dynamic light scattering S36 XII. Structural characterizations by atomic force microscopy S42 XIII. Structural characterizations by transmission electron microscopy S62 XIV. Nile Red encapsulation in cage with HE6-DNA S66 XV. Study of stability and assembly cooperativity by thermal denaturation S71 XVI. Titration of DNA-polymer conjugates to cube scaffold S75 XVII. Stepwise assembly of C4 with HEn-DNA S77 XVIII. Effect of the cage geometry on the assembly of higher-order structures S81 XIX. Isolation of the higher-order C4/HE6-DNA structures from the mixture S84 XX. Effect of concentration on the stability of the assemblies S86 XXI. References S88

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Page 1: Synergy of Two Assembly Languages in DNA Nanostructures

S1

Synergy of Two Assembly Languages in DNA Nanostructures: Self-Assembly

of Sequence-Defined Polymers on DNA Cages

Pongphak Chidchob, Thomas G. W. Edwardson, Christopher J. Serpell, & Hanadi F. Sleiman*

Department of Chemistry and Centre for Self-Assembled Chemical Structures (CSACS-CRMAA), McGill

University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada

Supplementary Information

Contents

I. General S2

II. Instrumentation S2

III. Solid-phase synthesis and purification S3

IV. DNA sequences and characterization S4

V. Cage design and assembly S13

VI. Cage assembly with HEn-DNA S15

VII. Determination of cage aggregation numbers S20

VIII. Gold nanoparticle labeling on C4/HE6-DNA S23

IX. Cage assembly with HE/HEG-DNA S28

X. Assembly characterization on agarose gel electrophoresis S33

XI. Particle size distributions by dynamic light scattering S36

XII. Structural characterizations by atomic force microscopy S42

XIII. Structural characterizations by transmission electron microscopy S62

XIV. Nile Red encapsulation in cage with HE6-DNA S66

XV. Study of stability and assembly cooperativity by thermal denaturation S71

XVI. Titration of DNA-polymer conjugates to cube scaffold S75

XVII. Stepwise assembly of C4 with HEn-DNA S77

XVIII. Effect of the cage geometry on the assembly of higher-order structures S81

XIX. Isolation of the higher-order C4/HE6-DNA structures from the mixture S84

XX. Effect of concentration on the stability of the assemblies S86

XXI. References S88

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I. General

Tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetate (EDTA), urea, 40%

acrylamide/bis-acrylamide (19:1), ammonium persulfate (APS), N,N,N',N'-tetramethylethane-1,2-diamine

(TEMED) and agarose were purchased from BioShop Canada Inc and used without further purification.

Acetic acid, ammonium hydroxide and boric acid were used as received from Fisher Scientific. GeneRuler

DNA Ladder Mix (cat.# SM1173) and DNA Gel Loading Dye (6X) were obtained from Thermos Scientific.

1000 Å 1 µmole universal synthesis column, reagents used for automated DNA synthesis and Sephadex G-

25 (super fine DNA grade) were purchased from BioAutomation. DMT-hexaethyloxy-glycol (HEG, cat.#

CLP-9765) and DMT-1,12-dodecane-diol (HE, cat.# CLP-1114) phosphoramidites were purchased from

ChemGenes Corporated. GelRed nucleic acid stain (10,000x in water) was obtained from Biotium Inc. Nile

Red was purchased from Sigma-Aldrich. 1xTBE buffer is composed of 90 mM Tris, 90 mM boric acid and

2 mM EDTA with a pH ~8.3. 1xTAMg buffer is composed of 45 mM Tris, 20 mM acetic acid and 12.5

mM MgCl2·6H2O, and its pH was adjusted to ~8.0 using glacial acetic acid.

II. Instrumentation

All standard DNA oligonucleotides were synthesized on solid supports using BioAutomation

MerMade MM6 DNA synthesizer. DNA quantification was performed by NanoDrop Lite

spectrophotometer (Thermo Scientific). Eppendorf Mastercycler 96-well thermocycler and Bio-Rad

T100TM thermal cycler were used to anneal all DNA structures. Polyacrylamide gel electrophoresis (PAGE)

was performed using 20x20 cm vertical Hoefer 600 electrophoresis units. Owl Mini gel electrophoresis unit

was used to perform agarose gel electrophoresis (AGE). HPLC purification was carried out on Agilent

Infinity 1260. Gels were imaged by BioRad ChemiDoc MP. LC-ESI-MS data were obtained on Dionex

Ultimate 3000 coupled to Bruker MaXis ImpactTM QTOF. Fluorescence data were measured by BioTek

Synergy H4 Hybrid Multi-Mode Microplate Reader. Melting profiles of DNA structures were monitored

by Cary 300 UV-Vis spectrophotometer equipped with Cary temperature controller (Agilent Technology).

Multimode 8 scanning probe microscope and Nanoscope V controller (Bruker, Santa Barbara, CA) was

used to acquire AFM images. DynaPro (model MS) molecular-sizing instrument was used to measure the

particle size distributions. TEM micrographs were acquired on FEI Tecnai 120 kV 12 microscope (FEI

electron optics).

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III. Solid-phase synthesis and purification

DNA synthesis was performed on a 1 µmole scale on a universal 1000 Å CPG solid support by

using standard method. Briefly, a phosphoramidite was activated by 0.25 M 5-(ethyl)-1H-thiotetrazole in

acetonitrile and coupled to DNA chains on the solid support. Failed coupling was capped by

THF/lutidine/acetic anhydride and 16% 1-methylimidazole/THF. Phosphorus (III) was oxidized to

phosphorus (V) with 0.02 M I2 in THF/pyridine/H2O. Coupling efficiency was monitored after the removal

of dimethoxytrityl (DMT) 5’-OH protecting groups by 3% dichloroacetic acid in dichloromethane. For,

non-nucleoside phosphoramidites, DMT-hexaethyloxy-glycol phosphoramidite and DMT-1,12-dodecane-

diol phosphoramidite were dissolved in acetonitrile to obtain 0.1 M solution under a nitrogen atmosphere

in a glove box, and added on the DNA synthesizer. The coupling time was extended to 5 minutes. After the

synthesis, the strand was deprotected and cleaved from the solid support by 28% aqueous ammonium

hydroxide for 16 hours at 60oC. The crude product was isolated, dried, and re-suspended in 1:1 H2O/8M

urea before loading to polyacrylamide/urea gel (12% for cage components and 15% for DNA-polymer

conjugates). The gel was run at 250 V for 30 minutes followed by 500 V for 45-60 minutes with 1xTBE as

the running buffer. The gel was then imaged and excised on TLC plate under a UV lamp. DNA was

extracted from the excised gel slabs by crushing and soaking in 11-12 mL Milli-Q water at 60oC overnight.

The solution was dried to approximately 1 mL before loading to Sephadex G-25 column. The purified DNA

was quantified by the absorbance at 260 nm.

Alternatively, the crude product of DNA-polymer conjugates was directly purified by reverse-

phase HPLC (Hamilton PRP-C18 5 µm 100 Å 2.1x150 mm). The sample was filtered using centrifuge tube

filter with 0.22 µm cellulose-acetate membrane, after which 0.5-0.75 OD260 of sample in Milli-Q water was

injected to the RP-HPLC. Two mobile phases were 50 mM triethylammonium acetate (TEAA, pH 8.0) and

HPLC grade acetonitrile with elution gradient of 3-50% acetonitrile over 30 minutes at 60oC. Detection

was carried out using a diode-array detector, monitoring the absorbance at 260 nm.

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IV. DNA sequences and characterization

The sequences of “DNA clips” necessary for cage assembly are listed in Supplementary Table 1.

The purity of the strands was evaluated by 12% denaturing PAGE (Supplementary Fig. 1). Approximately

0.02 nmole of strands was loaded on the gels. The gel was run at 250 V for 30 minutes then 500 V for 60

minutes with 1xTBE as the running buffer. The strands were further analyzed by LC-ESI-MS in negative

ESI mode. The results are summarized in Supplementary Table 2.

Supplementary Table 1. Sequences of the DNA clips (6 = HEG).

Strand Sequence (5’ 3’)

1AB TCGCTGAGTA 6 TCCTATATGGTCAACTGCTC 6 GCAAGTGTGGGCACGCACAC

6 GTAGTAATACCAGATGGAGT 6 CACAAATCTG

2AC CTATCGGTAG 6 TCCTATATGGTCAACTGCTC 6 TACTCAGCGACAGATTTGTG

6 GTAGTAATACCAGATGGAGT 6 CAACTAGCGG

3AD CACTGGTCAG 6 TCCTATATGGTCAACTGCTC 6 CTACCGATAGCCGCTAGTTG

6 GTAGTAATACCAGATGGAGT 6 GGTTTGCTGA

4AE CCACACTTGC 6 TCCTATATGGTCAACTGCTC 6 CTGACCAGTGTCAGCAAACC

6 GTAGTAATACCAGATGGAGT 6 GTGTGCGTGC

TP3-AB CCACACTTGC 6 TCCTATATGGTCAACTGCTC 6 CTACCGATAGCCGCTAGTTG

6 GTAGTAATACCAGATGGAGT 6 GTGTGCGTGC

PP4-AB TACCGGATCG 6 TCCTATATGGTCAACTGCTC 6 CTGACCAGTGTCAGCAAACC

6 GTAGTAATACCAGATGGAGT 6 CCGTAATTGC

PP5-AB CCACACTTGC 6 TCCTATATGGTCAACTGCTC 6 CGATCCGGTAGCAATTACGG

6 GTAGTAATACCAGATGGAGT 6 GTGTGCGTGC

1AA TCGCTGAGTA 6 TCCTATATGGTCAACTGCTC 6 GCAAGTGTGGGCACGCACAC

6 TCCTATATGGTCAACTGCTC 6 CACAAATCTG

2AA CTATCGGTAG 6 TCCTATATGGTCAACTGCTC 6 TACTCAGCGACAGATTTGTG

6 TCCTATATGGTCAACTGCTC 6 CAACTAGCGG

3AA CACTGGTCAG 6 TCCTATATGGTCAACTGCTC 6 CTACCGATAGCCGCTAGTTG

6 TCCTATATGGTCAACTGCTC 6 GGTTTGCTGA

4AA CCACACTTGC 6 TCCTATATGGTCAACTGCTC 6 CTGACCAGTGTCAGCAAACC

6 TCCTATATGGTCAACTGCTC 6 GTGTGCGTGC

Page 5: Synergy of Two Assembly Languages in DNA Nanostructures

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Strand Sequence (5’ 3’)

TP3-AA CCACACTTGC 6 TCCTATATGGTCAACTGCTC 6 CTACCGATAGCCGCTAGTTG

6 TCCTATATGGTCAACTGCTC 6 GTGTGCGTGC

PP4-AA TACCGGATCG 6 TCCTATATGGTCAACTGCTC 6 CTGACCAGTGTCAGCAAACC

6 TCCTATATGGTCAACTGCTC 6 CCGTAATTGC

PP5-AA CCACACTTGC 6 TCCTATATGGTCAACTGCTC 6 CGATCCGGTAGCAATTACGG

6 TCCTATATGGTCAACTGCTC 6 GTGTGCGTGC

Supplementary Figure 1. Gel electrophoresis assay for the DNA clips (12% denaturing PAGE with urea)

Lane 1: 1AB; lane 2: 2AC; lane 3: 3AD; lane 4: 4AE; lane 5: TP3-AB; lane 6: PP4-AB; lane 7: PP5-AB;

lane 8: 1AA; lane 9: 2AA; lane 10: 3AA; lane 11: 4AA; lane 12: TP3-AA; lane 13: PP4-AA; lane 14: PP5-

AA.

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Supplementary Table 2. Calculated and experimental masses in g/mole of the DNA clips obtained from

LC-ESI-MS.

Strand Calculated mass Experimental mass

1AB 26063.61 26064.3699

2AC 26038.60 26038.6906

3AD 25953.46 25953.5017

4AE 25960.53 25960.5960

TP3-AB 25988.52 25988.4558

PP4-AB 25974.55 25974.4902

PP5-AB 26037.56 26037.4980

1AA 25901.49 25901.2963

2AA 25922.50 25922.2974

3AA 25905.46 25905.2799

4AA 25804.40 25804.2342

TP3-AA 25826.40 25826.4197

PP4-AA 25812.42 25812.3707

PP5-AA 25875.44 25875.4066

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The sequences of DNA-polymer conjugates are listed in Supplementary Table 3. The purity of the

strands was evaluated by 15% denaturing PAGE with urea (Supplementary Fig. 2). The electrophoretic

mobility of HEn-DNA decreased when increasing number of HE repeats from 1 to 12 units, (lane 2-13). For

the strands with HEG repeats, their electrophoretic mobility decreased in more extent than those containing

only HE repeats (i.e. HE6-DNA in lane 7 and HE6-HEG6-DNA in lane 17). For the strands containing a

constant number of 6 HE and 6 HEG repeats per chain, their electrophoretic mobility increased with the

length of hydrophobic HE block (i.e. (HE-HEG)6-DNA in lane 14 and (HE3-HEG3)2-DNA in lane 16).

These suggest that the HE chains extend in solution in lesser extent than the HEG chains or, in other words,

the HE chains could have certain degree of chain folding. The strands were further analyzed by LC-ESI-

MS in negative ESI mode. The results are summarized in Supplementary Table 4.

Supplementary Table 3. Sequences of the polymer-DNA conjugates (6 = HEG, X = HE).

Strand Sequence (5’3’)

DNA TTTTTCAGTTGACCATATA

HE1-DNA XTTTTTCAGTTGACCATATA

HE2-DNA XXTTTTTCAGTTGACCATATA

HE3-DNA XXXTTTTTCAGTTGACCATATA

HE4-DNA XXXXTTTTTCAGTTGACCATATA

HE5-DNA XXXXXTTTTTCAGTTGACCATATA

HE6-DNA XXXXXXTTTTTCAGTTGACCATATA

HE7-DNA XXXXXXXTTTTTCAGTTGACCATATA

HE8-DNA XXXXXXXXTTTTTCAGTTGACCATATA

HE9-NDA XXXXXXXXXTTTTTCAGTTGACCATATA

HE10-DNA XXXXXXXXXXTTTTTCAGTTGACCATATA

HE11-DNA XXXXXXXXXXXTTTTTCAGTTGACCATATA

HE12-DNA XXXXXXXXXXXXTTTTTCAGTTGACCATATA

(HE-HEG)6-DNA X6X6X6X6X6X6TTTTTCAGTTGACCATATA

(HE2-HEG2)3-DNA XX66XX66XX66TTTTTCAGTTGACCATATA

(HE3-HEG3)2-DNA XXX666XXX666TTTTTCAGTTGACCATATA

HE6-HEG6-DNA XXXXXX666666TTTTTCAGTTGACCATATA

HEG6-HE6-DNA 666666XXXXXXTTTTTCAGTTGACCATATA

HE6-HEG12-DNA XXXXXX666666666666TTTTTCAGTTGACCATATA

HE12-HEG6-DNA XXXXXXXXXXXX666666TTTTTCAGTTGACCATATA

HE12-HEG12-DNA XXXXXXXXXXXX666666666666TTTTTCAGTTGACCATATA

Page 8: Synergy of Two Assembly Languages in DNA Nanostructures

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Supplementary Figure 2. Gel electrophoresis assay for the DNA-polymer conjugates (15% denaturing

PAGE with urea). Lane 1: DNA; lane 2-13: HEn-DNA (n=1-12); lane 14: (HE-HEG)6-DNA; lane 15: (HE2-

HEG2)3-DNA; lane 16: (HE3-HEG3)2-DNA; lane 17: HE6-HEG6-DNA; lane 18: HEG6-HE6-DNA; lane 19:

HE6-HEG12-DNA; lane 20: HE12-HEG6-DNA; lane 21: HE12-HEG12-DNA.

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Supplementary Table 4. Calculated and experimental masses of DNA-polymer conjugates in g/mole

obtained from LC-ESI-MS.

Strand Calculated mass Experimental mass

DNA 5764.99 5765.0000

HE1-DNA 6029.14 6029.1250

HE2-DNA 6293.29 6293.2188

HE3-DNA 6557.44 6557.4063

HE4-DNA 6821.59 6821.5000

HE5-DNA 7085.74 7085.6875

HE6-DNA 7349.89 7349.7813

HE7-DNA 7614.03 7613.8672

HE8-DNA 7878.18 7878.1250

HE9-DNA 8142.33 8142.2656

HE10-DNA 8406.48 8406.4063

HE11-DNA 8670.63 8670.4297

HE12-DNA 8934.78 8934.7778

(HE-HEG)6-DNA 9414.63 9414.5000

(HE2-HEG2)3-DNA 9414.63 9414.5000

(HE3-HEG3)2-DNA 9414.63 9414.5000

HE6-HEG6-DNA 9414.63 9414.5000

HEG6-HE6-DNA 9414.63 9414.5000

HE6-HEG12-DNA 11479.37 11484.5477

HE12-HEG6-DNA 10999.52 11004.7412

HE12-HEG12-DNA 13064.26 13070.0023

Page 10: Synergy of Two Assembly Languages in DNA Nanostructures

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The DNA-polymer conjugates were purified by RP-HPLC. All samples were run using the same

gradient of 3-50% acetonitrile in order to compare their relative hydrophobicity. For HEn-DNA, the

retention time increased with the number of HE repeats. In cases of the strands with a constant number of

6 HE and 6 HEG repeats per chain, their retention time increased with the length of hydrophobic HE block

(Supplementary Fig. 3), suggesting the increase in hydrophobicity of the polymer chain. With the same

number of HE repeats, the addition of HEG repeats resulted in lower retention time (Supplementary Fig.

4), indicating an increase in hydrophilicity as expected. Supplementary Table 5 shows the retention times

of all DNA-polymer conjugates used in this study.

Supplementary Table 5. Retention time of the DNA-polymer conjugates obtained from RP-HPLC.

Strand Retention time (minutes)

HE1-DNA 14.677

HE2-DNA 18.624

HE3-DNA 20.855

HE4-DNA 22.490

HE5-DNA 23.398

HE6-DNA 24.154

HE7-DNA 24.936

HE8-DNA 25.289

HE9-DNA 25.947

HE10-DNA 26.302

HE11-DNA 26.962

HE12-DNA 27.232

(HE-HEG)6-DNA 22.237

(HE2-HEG2)3-DNA 23.102

(HE3-HEG3)2-DNA 23.632

HE6-HEG6-DNA 24.399

HEG6-HE6-DNA 22.991

HE6-HEG12-DNA 23.618

HE12-HEG6-DNA 27.117

HE12-HEG12-DNA 26.838

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Supplementary Figure 3. HPLC chromatograms measured at 260 nm of the crude products of HEn-AT

(n=1-12). A gradient of 3-50% acetonitrile over 30 minutes was used.

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Supplementary Figure 4. HPLC chromatograms measured at 260 nm of the crude products of DNA-

polymer conjugates containing HE and HEG repeats. A gradient of 3-50% acetonitrile over 30 minutes was

used.

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V. Cage design and assembly

To assemble a cage, the equimolar amounts (1.25 pmole) of all required DNA clips were mixed in

1xTAMg buffer (10 µL) to obtain final cage concentration of 125 nM. The sample was heated at 95oC for

5 minutes, at 80oC for 3 minutes, cooled to 60oC (2 min/oC), and slowly cooled to 4oC (3 min/oC).

Supplementary Table 6 summarizes the combination of DNA clips for the construction of different cages.

Supplementary Table 6. DNA clip combinations for the construction of different cages.

Cage Clip strands Number of binding sites

TP3 1AB, 2AC, TP3-AB 3

C4 1AB, 2AC, 3AC, 4AD 4

PP5 1AB, 2AC, 3AC, PP4-AB, PP5-AB 5

TP6 1AA, 2AA, TP3-AA 6

C8 1AA, 2AA, 3AA, 4AA 8

PP10 1AA, 2AA, 3AA, PP4-AA, PP5-AA 10

An assembly of the cage was followed by native PAGE. The mixture of 10 µL of sample and 2 µL

of glycerol mix (7:1 glycerol/H2O) was loaded on 6% native PAGE with 1xTAMg as the running buffer.

The gel was run at 250 V for 2.5 hours and stained with GelRed. In Supplementary Fig. 5, all cages formed

in near-quantitative yields only in the presence of all required DNA clips (TP3 in lane 9, C4 in lane 11 and

PP5 in lane 13).

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Supplementary Figure 5. Stepwise assembly of three cages (6% native PAGE). Lane 1: 1AB; lane 2: 2AC;

lane 3: 3AD; lane 4: 4AE; lane 5: TP3-AB; lane 6: PP4-AB; lane 7: PP5-AB; lane 8: 1AB+2AC; lane 9:

1AB+2AC+TP3-AB (TP3); lane 10: 1AB+2AC+3AD; lane 11: 1AB+2AC+3AD+4AE (C4); lane 12:

1AB+2AC+3AD+PP4-AB; lane 13: 1AB+2AC+3AD+PP4-AB+PP5-AB (PP5).

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VI. Cage assembly with HEn-DNA

All required DNA clips and DNA-polymer conjugates in appropriate ratios (1.5 stoichiometric

equivalent per binding site) were mixed in 1xTAMg (10 µL) to obtain final cage concentration of 125 nM.

The final concentrations of DNA-polymer conjugates were 562.5, 750, 937.5, 1125, 1500 and 1875 nM for

TP3, C4, PP5, TP6, C8, PP10, respectively. Excess DNA-polymer conjugates were added to ensure complete

hybridization of the cages to the DNA-polymer conjugates. The sample was subjected to the same annealing

protocol as described in section V, mixed with 2 µL glycerol mix and then analyzed on 5% native PAGE

with 1xTAMg as the running buffer (250 V for 2.5hours).

In Supplementary Fig. 6 and 7, one face of trigonal prism TP3 and pentagonal prism PP5 was

decorated with 3 and 5 HEn-DNA (n=1-12), respectively. The quantization of the assembly was observed

similarly to cube C4/HEn-DNA as discussed in the article. Two major differences between three cage

systems decorated with hydrophobic polymers are: 1) a range of cage aggregation number for a given length

of polymers, and 2) a product distribution. In case of HE6-DNA, there were 3, 2 and 2 possible products for

TP3/HE6-DNA, C4/HE6-DNA and PP5/HE6-DNA, respectively. For cage dimers, they formed starting from

HE5-DNA to HE9-DNA for TP3 and C4, but only starting from HE5-DNA to HE7-DNA for PP5.

Page 16: Synergy of Two Assembly Languages in DNA Nanostructures

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Supplementary Figure 6. TP3 assembly with HEn-DNA (5% native PAGE). TP monomers were observed

from HE1-DNA to HE4-DNA. The higher-order structures were the major products for longer HE chains.

The bands corresponding to small higher-order structures such as dimer, trimer, and tetramer could be

clearly observed from TP3/HE5-DNA to TP3/HE9-DNA.

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Supplementary Figure 7. PP5 assembly with HEn-DNA (5% native PAGE). PP monomers were observed

from HE1-DNA to HE4-DNA. The higher-order structures were the major products for longer HE chains.

The bands corresponding to small higher-order structures such as dimer, trimer, and tetramer could be

clearly observed from PP5/HE5-DNA to PP5/HE9-DNA.

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Supplementary Fig. 8 and 9 show the assembly of TP6 and PP10 with HEn-DNA, respectively.

Folding of hydrophobic polymers of short to medium lengths inside the cages was observed in a similar

manner to cube C8/HEn-DNA. Long polymers generated higher-order structures for all types of cages.

Supplementary Figure 8. TP6 assembly with HEn-DNA (5% native PAGE). TP monomers were observed

from HE1-DNA to HE7-DNA. The electrophoretic mobility increased and remained constant from HE4-

DNA to HE7-DNA, suggesting the scaffold compaction due to the collapse of the hydrophobic HE chains

inside the interior of TP6. The higher-order structures were the main products for longer HE chains (HE6-

DNA to HE12-DNA).

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Supplementary Figure 9. PP10 assembly with HEn-DNA on 5% native PAGE. PP monomers were

observed from HE1 to HE8. The electrophoretic mobility increased and remained constant from HE5-DNA

to HE8-DNA, suggesting the scaffold compaction due to the collapse of the hydrophobic HE chains inside

the interior of PP10, similar to the cases of TP6/HEn-DNA and C8/HEn-DNA. The higher-order structures

were the main products from HE8-DNA to HE12-DNA.

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VII. Determination of cage aggregation numbers

To improve the resolution of higher-order bands on native PAGE, all samples were assembled

using the protocol described in section VI and were characterized by 3.5% native PAGE instead of 5%

native PAGE. In Supplementary Fig. 10, the first band of C4/HE6-DNA (lane 7, from the DNA ladder) was

assigned as the cube dimer. By comparing a position of this band to the DNA ladder on the left, the size of

the structure was estimated to be approximately 420 bp. Assuming that one polymer-decorated cube can

move approximately by 210 bp through the gel, the number of cubes for each higher-order band can be

estimated and summarized in Supplementary Table 7.

Supplementary Figure 10. TP3, C4 and PP5 assembly with HE6-8-DNA on 5% native PAGE.

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It should be noted that the smaller higher-order structures such as a cube dimer might be more

compact than the larger higher-order structures such as a cube pentamer possibly due to less electrostatic

repulsion between DNA nanostructures surrounding the hydrophobic core. Thus, it is also possible to use

the cube monomer, which normally appear at 250 bp based on other PAGE analysis, to predict cube

aggregation numbers. Assuming that one cube component will give ~250 bp for the larger higher-order

structures, the higher-order bands after the cube dimer will constitute of 2.6 and 4.2 cubes.

Supplementary Table 7. Cube aggregation numbers predicted from gel electrophoresis analysis.

Band Size (bp) 250 bp estimationa 210 bp estimationb

1 420 1.7 2.0

2 640 2.6 3.0

3 1050 4.2 5.0

4 1320 5.3 6.3

a estimated from the electrophoretic mobility of the cube monomer.

b estimated from the half value of the electrophoretic mobility of the cube dimer.

With the approximation from the electrophoretic mobility of the cage monomer, one polymer-

decorated cage will appear at 200 and 350 bp for TP3 and PP5, respectively. The cage aggregation number

estimated from these approximation are summarized in Supplementary Table 8.

As an alternative comparison, the dimer bands of TP3 and PP5 appeared at approximately 340 and

520 bp. Thus, the electrophoretic mobility of one decorated cage approximated from the cage dimers will

be 170 and 260 bp for TP3 and PP5, respectively. With the similar calculation to C4, cage aggregation

numbers of TP3 and PP5 with HE6-8-DNA could be predicted and summarized in Supplementary Table 8.

Page 22: Synergy of Two Assembly Languages in DNA Nanostructures

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Supplementary Table 8. Cage aggregation numbers predicted from gel electrophoresis analysis.

TP3 band Size (bp) 200 bp estimationa 170 bp estimationb

1 340 1.7 2.0

2 530 2.7 3.1

3 800 4.0 4.7

PP5 band Size (bp) 350 bp estimationa 260 bp estimationb

1 520 1.5 2.0

2 900 2.6 3.5

3 1400 4.0 5.4

4 1750 5.0 6.7

a estimated from the electrophoretic mobility of the cage monomer.

b estimated from the half value of the electrophoretic mobility of the cage dimer.

The estimation of cage aggregation number by comparing to the electrophoretic mobility of cage

monomers seems to be more consistent for all three types of cages. Therefore, we believe that increment

by one (such as dimer, trimer, tetramer, etc.) would be more likely to represent the choice for this assembly

process.

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VIII. Gold nanoparticle labeling on C4/HE6-DNA

As an alternative technique, the structural identity of C4/HE6-DNA was characterized by TEM. Due

to the low contrast of DNA, the cube was tagged with gold nanoparticle (AuNP) to increase the imaging

contrast. The cube aggregation number can be inferred from the number of AuNP observed in a close

proximity. The preparation of AuNP-CAB monoconjugates was performed according to the protocol

developed by Edwardson et al1 (Supplementary Fig. 11).

Supplementary Figure 11. Schematic of the preparation of AuNP-CAB monoconjugates.

The DNA strand (SBIS) containing two cyclic disulfide moieties at its 5’end was decorated on B

face of the cube CAB, which contains 4 A edges on one face and 4 B edges on another face. This cube CAB

can be made from the clips 1AB, 2AB, 3AB and 4AB. Attachment of AuNP to the cube is driven by the

formation of Au-S bonds between AuNP and SBIS. The sequences of all DNA strands for this experiments

are listed in Supplementary Table 9.

Supplementary Table 9. Sequences of DNA clips for CAB and SBIS strand (6 = HEG, X = HE, B =

symmetrical branching, S = cyclic dithiol).

Strand Sequence (5’ 3’)

1AB TCGCTGAGTA 6 TCCTATATGGTCAACTGCTC 6 GCAAGTGTGGGCACGCACAC

6 GTAGTAATACCAGATGGAGT 6 CACAAATCTG

2AB CTATCGGTAG 6 TCCTATATGGTCAACTGCTC 6 TACTCAGCGACAGATTTGTG

6 GTAGTAATACCAGATGGAGT 6 CAACTAGCGG

3AB CACTGGTCAG 6 TCCTATATGGTCAACTGCTC 6 CTACCGATAGCCGCTAGTTG

6 GTAGTAATACCAGATGGAGT 6 GGTTTGCTGA

4AB CCACACTTGC 6 TCCTATATGGTCAACTGCTC 6 CTGACCAGTGTCAGCAAACC

6 GTAGTAATACCAGATGGAGT 6 GTGTGCGTGC

SBIS SXB TTTTACCATCTGGTATTAC

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To assemble the cube CAB with SBIS, 1.25 µM (100 µL) of CAB was mixed with SBIS (31.25 µL of

5 µM SBIS, 1.25 equivalent) in 1xTBE supplemented with 150 mM NaCl (TBEN). The samples were

annealed from 95oC to 4oC over 6 hours. To 50 µL solution of 1 µM BSPP-coated 10-nm AuNP in 1xTBEN

was added 90.1 µL of 555 nM CAB-SBIS (1:1 ratio) and 16 µL of 10 mg/mL BSPP in 1xTBEN to obtain

final BSPP concentration of 1 mg/mL. After incubation at room temperature overnight, 5 µL of 0.2 M

HOOC-PEG8-S-S-PEG8-COOH in 1xTBEN was added to the mixture and leaved at room temperature for

30 minutes. The crude mixture was then loaded on 3% agarose gel and the gel was run at 80 V for 1 hour

with 1xTBE as the running buffer. The band corresponding to AuNP-CAB monoconjugates was excised

(Supplementary Fig. 12, upper band in lane 1), cut into small pieces and soaked in 1xTAMg at 4oC. After

1-2 days, the liquid was isolated from the gel slices and centrifuged at 12000xg for 30 minutes in the cold

room to collect the AuNP-CAB. As a control, unbound AuNPs were also isolated using the same method.

The purity of the isolated products were evaluated by 3% AGE. In lane 3 of Supplementary Fig.

12, there were two bands for the AuNP-CAB monoconjugates. The upper band was the target structure and

the lower band could be AuNP-SBIS without CAB (AuNP-SBIS) as this band showed lower electrophoretic

mobility than free AuNPs in lane 2. The concentration of AuNP-CAB was quantified by the absorption of

AuNP at 450 nm using the extinction coefficient of 10-nm AuNP (𝜀450 nm = 6.15x107 M-1cm-1) reported in

the literature.2 The AuNP-CAB was used for the next experiment without further purification.

Supplementary Figure 12. Agarose gel electrophoresis assay for AuNP-CAB monoconjugates preparation.

Lane 1: crude mixture; lane 2: free AuNPs isolated from the lower band in lane 1; and lane 3: AuNP-CAB

isolated from the upper band in lane 1. The upper band in lane 3 was the target AuNP-CAB while the lower

band was AuNP-SBIS4 that lost the cube CAB components during the extraction process.

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To assemble the higher-order structures, AuNP-CAB and HE6-DNA were mixed together in

1xTAMg and incubate at room temperature overnight. While the concentrations of AuNP and HE6-DNA

were kept at 125 and 750 nM, the actual concentration of AuNP-CAB was lower than 125 nM due to the

presence of AuNP-SBIS4. However, excess HE6-DNA will not interfere with the formation of higher-order

structures.3 HE6-DNA was also added to the isolated free AuNP as the control. The assembly products were

run on 3% AGE at 80 V for 1.5 hours with 1xTAMg as the running buffer. In Supplementary Fig. 13, there

was no change in the band of AuNP after addition of HE6-DNA (lane 1 and 2), suggesting little or no

interaction between HE6-DNA and AuNPs. In contrast, addition of HE6-DNA to AuNP-CAB resulted in the

smearing on the gel although most of the products remained as unbound structures (lane 3 and 4).

Supplementary Figure 13. Agarose gel electrophoresis assay for the assembly of AuNP and AuNP-CAB

with HE6-DNA. While there was no change after addition of HE6-DNA in AuNP, the smear could be seen

in case of AuNP-CAB with HE6-DNA.

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The structures were further characterized by TEM. For the sample preparation, 5 µL of 500 µg/mL

bacitracin was deposited on carbon-coated Cu grid then wicked off by using filter paper after 1 minute.

Then the grid was washed with 5 µL of water then wicked off excess. The sample was diluted 6x with

1xTAMg, then 5 µL was deposited on the grid and washed with 5 µL of water before drying under vacuum

for 4 hours. TEM micrographs of AuNP/HE6-DNA and AuNP-CAB/HE6-DNA are shown in Supplementary

Fig. 14 and 15, respectively.

Supplementary Figure 14. TEM images of AuNP-CAB.

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Supplementary Figure 15. TEM images of AuNP-CAB with HE6-DNA. The clusters of 2-4 AuNPs in a

close proximity can be observed. (2 AuNPs = 12.9%, 3 AuNPs = 5.6%, 4 AuNPs = 0.7%)

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IX. Cage assembly with HE/HEG-DNA

All samples were assembled using the protocol described in section VI and were characterized by

native PAGE. In Supplementary Fig. 16 and 17, trigonal prism TP and pentagonal prism PP were assembled

with HE/HEG-DNA containing a constant 6 HE and 6 HEG repeats but of different sequences. All DNA-

polymer conjugates resulted in monomeric structures with one exception. The electrophoretic mobility

increased with the length of HE blocks (i.e., lane i-iii). This suggests that longer HE blocks could have

some degree of chain folding which will make the assembly become less floppy in solution and move faster

through the gel. Only HE6-HEG6-DNA leaded to the formation of larger assemblies. Therefore, the

sequence order is also an important factor to determine the outcome of the assembly. With HEn-HEGm-

DNA (n,m=6,12), all types of cages showed higher-order structures (Supplementary Fig. 18-20).

Supplementary Figure 16. TP3 and TP6 assembly with HE/HEG-DNA (5% native PAGE). Only TP/HE6-

HEG6-DNA formed the higher-order structures, while the rest of the conjugates resulted in the monomeric

structure.

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Supplementary Figure 17. PP5 and PP10 assembly with HE/HEG-DNA (5% native PAGE). Only C/HE6-

HEG6-DNA formed the higher-order structures, while the rest of the conjugates resulted in the monomeric

structure.

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Supplementary Figure 18. TP3 and TP6 assembly with HE/HEG-DNA (5% native PAGE). All polymers

with trigonal prisms generated the higher-order structures.

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Supplementary Figure 19. C4 and C8 assembly with HE/HEG-DNA (5% native PAGE). All polymers

with cubes generated the higher-order structures.

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Supplementary Figure 20. PP5 and PP10 assembly with HE/HEG-DNA (5% native PAGE). All polymers

with pentagonal prisms generated the higher-order structures.

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X. Assembly characterization on agarose gel electrophoresis

Some of the assembled structures cannot be fully characterized by native PAGE as they remained

as nonpenetrating materials in the wells. Although this suggests that these structures are large aggregates,

agarose gel electrophoresis (AGE) was used in order to study size, electrophoretic mobility and number of

possible structures of these aggregates. First, a gel behavior of some DNA-polymer conjugates was

examined on 2.5% native AGE under native condition. These DNA-polymer amphiphiles were assembled

in 1xTAMg (10 µL) at 5 µM by using the same annealing protocol as described in Section V. The samples

were mixed with 2 µL of 6X loading dye before loading on the gel. The gel was run at 80 V for 2.5 hours

with 1xTAMg as the running buffer (Supplementary Fig. 21).

For HEn-DNA, their electrophoretic mobility decreased with the number of HE repeats.

Surprisingly, HE6-DNA (lane 4), which was reported to form smaller micelles than HE12-DNA,4 showed

lower electrophoretic mobility than HE12-DNA (lane 5). This reduction in electrophoretic mobility was also

observed in the block copolymers containing 6 HE repeats (lane 6 and 7), in comparison to the copolymers

containing 12 HE repeats (lane 8 and 9). While a factor that contributes to this unusual observation is

unknown, it is likely that HE chains of intermediate length might exhibit certain degree of flexibility of the

hydrophobic core. Considering that both HE6-DNA and HE12-DNA can form micelles in solution, HE chain

folding of less hydrophobic HE6-DNA might be less tight than that of HE12-DNA, allowing a “breathing”

of HE6 chains which reduced its electrophoretic mobility.

Supplementary Figure 21. Gel electrophoresis assay for the DNA-polymer conjugates (2.5% native AGE).

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The cube C8 with HEn-DNA were then analyzed on AGE. In Supplementary Fig. 22, the assemblies

of C8 with the polymer chains (n=3-6) remained as nonpenetrating materials in the wells. This observation

is consistent with the unusual behavior of HE6-DNA amphiphiles on AGE observed in Supplementary Fig.

21. However, we predicted that these assemblies should be able to migrate through the agarose gel due to

other structural evidences which conclude that they are cube monomers whose hydrophobic chains

collapsing inside the interior of the cubes. For C8 with longer HEn-DNA, nonpenetrating materials and a

smear were the major products.

Supplementary Figure 22. C8 assembly with HEn-DNA-DNA (1.5% native AGE).

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In the case of HE/HEG copolymers (Supplementary Fig. 23), C8 with HEn-HEGm-DNA resulted

nonpenetrating materials which are expected due to the aggregation products. However, C4/HE6-HEGm-

DNA (m=6,12) did not migrate through the gel. These structures were smaller than C4/HE12-HEGm-DNA

(m=6,12) as observed by DLS and AFM, so they should have comparable or higher electrophoretic mobility

when compared to C4/HE12-HEGm-DNA (m=6,12), which were able to migrate through the gel. Thus, it is

challenging to interpret the identity of the structure by using AGE technique, considering the unknown

interaction between HEn chains and agarose materials that might affect the interpretation.

Supplementary Figure 23. C4 and C8 assembly with HE/HEG-DNA (2.5% native AGE).

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XI. Particle size distributions by dynamic light scattering

20 µL of samples were analyzed on a DynaPro (model MS) molecular-sizing instrument using a

laser wavelength of 824 nm at 20oC or 25oC. Each sample was measured at least three times. Supplementary

Table 10 summarizes the hydrodynamic radii (Rh) and the polydispersity percentages of the assemblies of

C4 and the DNA-polymer conjugates. Supplementary Fig. 24 shows the histograms and the corresponding

correlation curves of all structures.

Supplementary Table 10. Hydrodynamic radii (Rh) and % polydispersity of C4 assemblies with DNA-

polymer conjugates obtained from DLS

Structure Rh (nm) % polydispersity

C4 5.4±0.3 17.2±7.4

C4/AT 6.0±0.6 19.1±9.8

C4/HE6-DNA 7.7±1.0 25.5±8.4

C4/HE7-DNA 11.0±1.6 25.0±12.6

C4/HE12-DNA 17.4±1.5 19.4±7.8

C4/HE6-HEG6-DNA 13.6±1.1 14.7±7.1

C4/HEG6-HE6-DNA 6.9±0.9 25.2±16.1

C4/HE6-HEG12-DNA 17.7±1.4 17.3±8.9

C4/HE12-HEG6-DNA 19.7±0.9 15.6±7.9

C4/HE12-HEG12-DNA 21.0±1.7 16.2±9.0

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Supplementary Figure 24. DLS results of C4 with DNA-polymer conjugates. Left: the histogram showing

the size distribution of the assemblies; right: the corresponding intensity correlation function.

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Supplementary Table 11 summarizes hydrodynamic radii (Rh) and polydispersity percentages of

the assemblies of C8 and the DNA-polymer conjugates. Although the polydispersity of the size distribution

was in general considerably narrow (<15%), it should be noted that the size of large structures, including

C8/HE12-DNA, C8/HE6-HEG12-DNA, C8/HE12-HE6-DNA and C8/HE12-HEG12-DNA, varied to some degree

from one measurement to another. As such, the Rh values for these structures might not truly represent the

actual size in solution. However, these values could be useful to provide their relative sizes. Supplementary

Fig. 25 shows the histograms and the corresponding correlation curves of all structures.

Supplementary Table 11. Hydrodynamic radii and % polydispersity of C8 assemblies with DNA-polymer

conjugates obtained from DLS

Structure Rh (nm) % polydispersity

C8 5.4±0.6 23.2±9.5

C8/DNA 7.1±0.6 16.3±7.3

C8/HE6-DNA 6.4±0.4 22.1±2.9

C8/HE8-DNA 32.6±1.8 32.6±2.0

C8/HE12-DNA 63.0±1.8 11.7±4.8

C8/HE6-HEG6-DNA 22.0±3.2 47.8±5.7

C8/HEG6-HE6-DNA 8.3±0.7 30.4±12.9

C8/HE6-HEG12-DNA 145.0±2.8 13.7±0.4

C8/HE12-HEG6-DNA 91.9±10.9 11.9±1.4

C8/HE12-HEG12-DNA 148.6±14.6 9.5±3.4

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Supplementary Figure 25. DLS results of C8 with DNA-polymer conjugates. Left: the histogram showing

the size distribution of the assemblies; right: the corresponding intensity correlation function.

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XII. Structural characterizations by atomic force microscopy

The sample was diluted with 1xTAMg from 125 nM to 41.7-62.5 nM with respect to the cages. 5

µL of sample was deposited on freshly cleaved mica for 5 seconds, and washed three times with 50 µL of

H2O. Excess liquid was blown off by the stream of nitrogen for 30 seconds. The sample was then dried

under vacuum for at least 20 minutes prior to imaging. Measurement was acquired in ScanAsyst mode

under dry condition using ScanAsyst-Air triangular silicon nitride probe (tip radius = 2 nm, k = 0.4 N/m, fo

= 70 kHz; Bruker, Camarillo, CA).

Images were processed by NanoScope Analysis 1.40 Software. The data was treated with flattening

to correct tilt, bow and scanner drift. Average particle sizes, heights and numbers of particles (N) were

obtained from Particle Analysis function, and edge lengths of some particles were measured by Section

function. Supplementary Fig. 26-31 show the AFM images of C4 with the DNA-polymer conjugates.

Supplementary Figure 26. Additional AFM images of C4/HE6-DNA. Elongated structures and triangular

structures could be clearly observed. Average size of the particles was 22.7±3.4 nm with height of 2.1±0.3

nm (N=171). The edge length of the structures was ~27-32 nm, which should account for approximately

two cubes. The percentages of cube dimer, trimer, and tetramer were 76%, 17% and 7%, respectively

(N=138). The cube tetramer was defined as the triangles with raising heights at the center.

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Supplementary Figure 27. AFM images of C4/HE7-DNA. Elongated structures and triangular structures

could be clearly observed. Average size of the particles was 23.3±5.6 nm with height of 2.3±0.4 nm

(N=840). The percentages of cube dimer, trimer, and tetramer were 52%, 24% and 24%, respectively

(N=321). The cube tetramer was defined as the triangles with raised heights at the center. It should be noted

that the percentage of cube dimers obtained from C4/HE7-DNA was lower than that of C4/HE6-DNA.

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Supplementary Figure 28. Additional AFM images of C4/HE6-HEG6-DNA. Polygonal rings with 3-5

vertices were the main products along with some of the chain-like structures. Average size of the particles

was 26.4±8.2 nm with height of 2.0±0.3 nm (N=178). The edge length of the closed polygon was ~30 nm.

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Supplementary Figure 29. Additional AFM images of C4/HE6-HEG12-DNA. Average size of the particles

was 25.4±10.0 nm with height of 2.1±0.4 nm (N=49). There were clusters of the spherical structures of ~12

nm, which could represent individual cube C4. The relative position between spherical structures within a

close proximity suggests that the assemblies could be similar to C4/HE6-HEG6-DNA. It is possible that 1)

longer HEG block provides more spacing, and 2) the assemblies breaks apart upon the deposition on mica

surface in some extent due to larger hydrophilic-to-hydrophobic content of the polymer. Therefore, it was

difficult to provide a precise yield from the AFM images, because of the presence of some misassembled

structures of unknown composition in the image background.

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Supplementary Figure 30. Additional AFM images of C4/HE12-HEG6-DNA. Well-defined spherical

structures of 53.9±8.8 nm were clearly observed (N=271). The height was 4.4±1.1 nm. Some structures

contained high features at the center of the sphere and some showed hollow features.

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Supplementary Figure 31. Additional AFM images of C4/HE12-HEG12-DNA. The doughnut-shaped ring

structures (size = 47.7±9.8 nm, height = 2.6±0.8 nm, N=353) were the main structures. They displayed

similar morphology to C4/HE6-HEG6-DNA but were bigger in size.

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Supplementary Fig. 32 shows the structures of C8 while the AFM characterizations of C8 assembly

with the DNA-polymer conjugates are shown in Supplementary Fig. 33-39.

Supplementary Figure 32. AFM images of C8. Spherical structures representing individual cube C8 were

the main products. The average size was 17.0±2.8 nm and the height was 1.8±0.2 nm (N=568).

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Supplementary Figure 33. AFM images of C8/HE6-DNA. Spherical structures with the size of 19.1±4.8

and height of 1.8±0.4 nm can be observed (N=236). This further supports that the assemblies formed cube

monomers.

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Supplementary Figure 34. AFM images of C8/HE8-DNA. Spherical features with the size of 29.6±7.4 nm

were mainly observed (N=251). The height was 2.6±0.8 nm.

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Supplementary Figure 35. AFM images of C8/HE12-DNA. The size of most heterogeneous particles was

within the range of 48.8±14.8 nm (height = 6.9±2.5 nm, N=135). Larger aggregates could be observed as

well, which could be aggregations of small particles.

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Supplementary Figure 36. AFM images of C8/HE6-HEG6-DNA. The size of most of the spherical

aggregates was 26.7±7.3 nm with the height of 2.2±0.2 nm (N=333). Larger aggregates of small spherical

structure such as chains and irregular structures can be observed as well but in smaller proportion.

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Supplementary Figure 37. AFM images of C8/HE6-HEG12-DNA. Relatively spherical structures with the

size of 46.3±9.7 nm (height = 2.2±0.4 nm, N=136) can be observed.

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Supplementary Figure 38. AFM images of C8/HE12-HEG6-DNA. Majority of the population contained

the spherical structures with the size of 45.6±10.7 nm and the height of 5.0±1.6 nm (N=86). Larger

aggregate can be observed and could be an aggregation of small spherical structures.

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Supplementary Figure 39. AFM images of C8/HE12-HEG12-DNA. Majority of the population contained

the spherical structures with the size of 50.7±7.2 nm (height = 2.8±0.6 nm, N=40).

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Supplementary Fig. 40 shows the structures of PP5 while the AFM characterizations of PP5

assembly with the DNA-polymer conjugates are shown in Supplementary Fig. 41-43.

Supplementary Figure 40. AFM images of PP5. Spherical features with the size of 20.8±6.0 nm (height =

1.6±0.2 nm, N=710) were clearly observed. These spheres can represent individual PP5.

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Supplementary Figure 41. AFM images of PP5/HE6-DNA. Most features were linear dimeric structures

(length of ~30 nm) with small population of the triangular structures. Average particle size was 24.4±3.8

nm with height of 5.3±7.9 nm (N=222).

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Supplementary Figure 42. AFM images of PP5/HE7-DNA. Triangular structures with edge length of ~27-

30 nm were mainly observed Average particle size was 25.8±4.4 nm with height of 2.4±0.4 nm (N=298).

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Supplementary Figure 43. AFM images of PP5/HE6-HEG6-DNA. Polygonal rings with edge length of

~24-27 nm which is in similar dimension to C4/HE6-HEG6-DNA were observed. Average size was 28.9±6.8

nm and height was 1.9±0.2 nm (N=58).

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Supplementary Fig. 44 and 45 show the AFM characterizations of TP3/HE7-DNA and TP3/HE6-

HEG6-DNA, respectively.

Supplementary Figure 44. AFM images of TP3/HE7-DNA. Triangular structures can be mostly observed.

There were some linear dimeric structures as well as quadrilateral structures. Average size was 21.2±4.5

nm and the height was 2.0±0.4 nm (N=243).

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Supplementary Figure 45. AFM images of TP3/HE6-HEG6-DNA. Polygonal rings similar to C4/HE6-

HEG6-DNA were observed. Average size was 27.8±8.4 nm and height is 2.2±0.3 nm (N=156).

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XIII. Structural characterizations by transmission Electron Microscopy

2 µL of sample was deposited on the carbon film coated 400-mesh copper grid (Electron

Microscopy Sciences, Hatfield, PA) for one minutes. Excess liquid was blotted off with an edge of a filter

paper. The sample was washed three times with 20 µL H2O and blotted with filter paper. The sample was

dried under vacuum at least 30 minutes prior to the imaging. Average particle sizes and numbers of particles

(N) were analyzed by ImageJ software.

Supplementary Figure 46. TEM images of C4/HE12-HEG6-DNA. Spherical structures (size=24.7±3.9 nm,

N=337) were clearly observed.

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Supplementary Figure 47. TEM images of C4/HE12-HEG12-DNA. Aggregates with the size of 30.3±5.4

nm were clearly observed (N=462).

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Supplementary Figure 48. TEM images of C8/HE12-HEG6-DNA. Two size distributions of the structures

were 107.5±28.5 nm for larger aggregates (N=124) and 22.1±3.9 nm for relatively homogeneous spherical

structures (N=282).

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Supplementary Figure 49. TEM images of C8/HE12-HEG12-DNA. Two size distributions of the structures

were 138.7±45.3 nm for larger aggregates (N=40) and 23.3±5.0 nm for relatively homogeneous spherical

structures (N=63).

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XIV. Nile Red encapsulation in cages with HE6-DNA

Cages (125 nM) and HE6-DNA or unmodified DNA (1.5 stoichiometric equivalent; 1.125, 1.5 and

1.875 µM for TP6, C8 and PP10, respectively) were annealed together in 1xTAMg. To prepare Nile Red for

an encapsulation, in a separated glass vial was added 50 µL of 1 mM Nile Red solution in acetone followed

by drying at room temperature to obtain dried film of Nile Red. Then, 400 µL of the assembled cage

solutions were added to the vials (final concentration of Nile Red = 125 µM or 1000x excess with respect

to cage concentration), mixed by a vortexer for 1 minute and gently shake in dark using the rotator for 19

hours. Excess Nile Red was removed by centrifugation at 13.4 krpm for 10 minutes in the cold room. After

that the samples were concentrated by 10k MWCO centrifugal devices at 13.4 krpm for 10 minutes in the

cold room. Supplementary Fig. 50 shows the schematic of this experiment.

Supplementary Figure 50. Encapsulation of Nile Red in cages with HE6-DNA or unmodified DNA.

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To determine the concentration of concentrated cages after removal of excess Nile Red, the cages

decorated with unmodified DNA or HE6-DNA were analyzed on 12% denaturing PAGE. By comparing to

the band intensity of known cage concentrations, the band intensity of concentrated cages can be converted

to the concentration by using the standard curve of cage concentration versus band intensity (Supplementary

Fig. 51). In addition, to confirm the cage integrity after Nile Red encapsulation, the concentrated cages

were analyzed on 5% native PAGE, where 2.5 µL of samples were mixed with 7.5 µL 1xTAMg and 2 µL

of glycerol mix, and then loaded on the gel. In Supplementary Fig. 52, the major products were the cage

monomers, indicating that all cages with either unmodified DNA or HE6-DNA remained intact after

removal of excess Nile Red.

Supplementary Figure 51. Nile Red encapsulation in the cage with unmodified DNA or HE6-DNA. Left:

12% denaturing PAGE for determination of the cage concentrations. Known cage concentrations from left

to right were 1, 0.5 and 0.25 µM. Right: the standard curves of cage concentrations constructed from the

band intensities.

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Supplementary Figure 52. Nile Red encapsulation in the cage with unmodified DNA or HE6-DNA (5%

native PAGE) for evaluating the cage integrity after Nile Red encapsulation and purification.

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To measure fluorescent signals of the encapsulated Nile Red, 20 µL of concentrated samples were

mixed with 80 µL acetone and transferred to 96 well-plate. The plate was read using the BioTek Synergy

well-plate fluorometer. The excitation was at 535 nm with a slit width of 9 nm and the emission was

monitored between 560 and 750 nm. A series of Nile Red of known concentrations was prepared to

construct the standard curve in order to determine the concentration of Nile Red encapsulated within the

cages (Supplementary Fig. 53). It should be noted that Nile Red fluorescent emission can be influenced by

the polarity of the surrounding environment. Thus, in this protocol, acetone was added to the samples prior

to the fluorescent measurement. Although DNA/DNA amphiphilic components in the sample might have a

possible effect on the fluorescence of Nile Red, Nile Red should mainly dissolve in the organic phase due

to its high solubility in acetone. With a constant volume ratio between the buffer and acetone, it is most

likely that Nile Red molecules in different samples sharing similar a solvent environment could behave

similarly. Thus, a calibration curve can be used to determine Nile Red concentrations in different cage

samples.

Supplementary Figure 53. Emission spectra of Nile Red encapsulated within cages with either unmodified

DNA or HE6-DNA and standard curve of Nile Red concentration used to determine concentration of Nile

Red within cages.

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The data for this experimental set was summarized in Supplementary Table 12.

Supplementary Table 12. Nile Red encapsulation summary of the data set from Supplementary Fig. 51-

53.

[NR], µM [cage], µM [NR]:[cage]

1xTAMg 0.231 0 -

TP6/DNA 0.235 0.302 0.8

TP6/HE6-DNA 0.345 0.340 1.0

C8/DNA 0.238 0.344 0.7

C8/HE6-DNA 1.577 0.404 3.9

PP10/DNA 0.253 0.453 0.6

PP10/HE6-DNA 4.503 0.431 10.4

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XV. Study of stability and assembly cooperativity by thermal denaturation

Cube (375 nM) and DNA-polymer conjugates (1.5 stoichiometric equivalence: 2.25 µM for C4 and

4.5 µM for C8) were mixed and annealed in 1xTAMg. Then, 100 µL of the samples were transferred to

quartz cuvettes and few drops of silicone oil were added on top. The absorbance at 260 nm in response to

a temperature change was monitored (Supplementary Fig. 54 left). The temperature was increased from

25oC to 95oC with 1oC increment per minute. The first derivatives of the normalized melting curves were

fitted with Lorenztian distribution function using OriginPro 2015 software. Then, the melting temperature

(Tm) was determined from the highest values of the first derivatives, and the full width half maximum

(fwhm) of the curves which can be used to indicate the degree of cooperativity in DNA binding5 was also

obtained (Supplementary Fig. 54 right). All Tm and fwhm values are listed in Supplementary Table 13.

Melting curves for all structures are shown in Supplementary Fig. 55.

Supplementary Figure 54. Representative example of melting curve and its first derivative curve.

Lorenztian distribution was used to fit the first derivative curve to obtain two parameters: the peak

maximum indicating the melting temperature (Tm) of DNA nanostructure and the full width half maximum

(fwhm) indicating the degree of cooperativity.

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Supplementary Table 13. Melting temperature of the cube scaffolds with the DNA-polymer conjugatesa

DNA-polymer conjugates C4 C8

Tm (oC) fwhm (oC) Tm (oC) fwhm (oC)

DNA 54.6±1.8 10.3±1.8 54.6±0.4 10.1±1.0

HE6-DNA 56.7±0.2 4.5±0.7 59.9±0.2 4.0±0.1

HE6-HEG6-DNA 57.1±0.2 3.8±0.3 62.8±0.7

(57.8±0.3,

62.6±0.6)

8.2±2.5

(4.1±1.6,

6.1±1.4)

HEG6-HE6-DNA 54.9±0.4 7.7±1.1 55.9±0.1 7.3±0.4

HE12-DNA 56.8±0.2 5.3±0.3 65.8±1.0 6.7±1.6

HE6-HEG12-DNA 56.4±0.3 4.2±0.5 61.9±0.8

(59.8±0.6,

64.7±0.4)

10.5±1.2

(5.7±0.6,

5.7±0.6)

HE12-HEG6-DNA 57.7±0.2 3.8±0.1 66.0±1.7

(63.0±0.5,

67.1±1.2)

7.6±1.3

(3.5±3.0,

5.8±0.4)

HE12-HEG12-DNA 57.6±0.3 3.7±0.4 66.3±1.0

(62.3±0.1,

66.9±0.5)

10.1±1.3

(6.6±0.2,

6.6±0.2)

a Some of the first derivative curves contained two local maxima. In addition to the values obtained from

the fitting of the global maximum of the curves, the numbers in the parentheses were obtained from the

multi peak fitting of the two local maxima.

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Supplementary Figure 55. Melting curves of C4 and C8 with the DNA-polymer conjugates

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XVI. Titration of DNA-polymer conjugates to cube scaffold

To examine whether hydrophobic interactions can improve the assembly cooperativity of DNA

cubes, C8 (125 nM) and DNA/HE6-DNA at various concentrations (500 nM, 750 nM, 1 µM 1.25 µM and

1.5 µM) were annealed in 1xTAMg (10 µL) then analyzed on 5% native PAGE. In Supplementary Fig. 56,

higher concentrations of both unmodified DNA and HE6-DNA promoted the formation of fully-bound

cubes. While at low (substoichiometric) concentrations of unmodified DNA and C8/DNA showed the band

ladder corresponding to C8 with one, two, three and more DNAs (intermediate structures), there were only

unbound C8 and fully-bound C8 in the case of C8/HE6-DNA, suggesting the cooperativity of the binding of

HE6-DNA to the C8 scaffolds.

Supplementary Figure 56. Titration of DNA and HE6-DNA to C8 (5% native PAGE). Left: C8 with DNA.

Right: C8 with HE6-DNA. The concentrations of DNA/HE6-DNA for one binding site were 0.5, 0.75, 1.0,

1.25 and 1.5 stoichiometric equivalences. All-or-non binding (unbound or fully-bound cubes) was observed

in C8 with HE6-DNA, suggesting the binding cooperativity induced by hydrophobic interactions.

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To further examine the hydrophobically driven assembly cooperativity, the cube C4 (125 nM) and

HE/HEG-DNA at various concentrations (500 nM, 750 nM and 1 µM) were annealed in 1xTAMg (10 µL)

then analyzed on 5% native PAGE. In all cases, higher concentrations of HE/HEG-DNA promoted the

formation of fully-bound cubes (Supplementary Fig. 57). However, the band ladder of C4 containing 1-4

strands of HE/HEG-DNA was observed, except for C4/HE6-HEG6-DNA. This suggests that the binding of

HE/HEG-DNA except HE6-HEG6-DNA to C4 is not cooperative. According to the study of these HE/HEG-

DNA amphiphiles by Edwardson et al., only HE6-HEG6-DNA was able to form the micelles in solution.4

This further suggests that the micellization of the polymers could be the significant contribution to the

observed cooperativity.

Supplementary Figure 57. Titration of HE/HEG-DNA to C4 (5% native PAGE). The concentrations of

HE/HEG-DNA for one binding site were 1, 1.5 and 2 stoichiometric equivalences, except for HE6-HEG6-

DNA where 1 and 2 equivalences were used. No all-or-none binding was evidenced for all HE/HEG-DNA

except HE6-HEG6-DNA, which generated no intermediate structure between unbound cube and fully-bound

cube.

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XVII. Stepwise assembly of C4 with HEn-DNA

C4 and HEn-DNA were separately prepared in 1xTAMg at 250 nM and 1.5 µM, respectively. Both

solutions were subjected to the same annealing protocol as described in section V. To 5 µL of C4 was added

5 µL of 1.5 µM HEn-DNA to obtain final C4 and HEn-DNA concentrations of 125 nM and 750 nM,

respectively. The mixtures were incubated in water bath at 37oC for 30 minutes then analyzed by native

PAGE. One-pot assembly of C4 with HE6-8-DNA were used as the controls. In Supplementary Fig. 58, the

stepwise assembly of C4 with HEn-DNA resulted in similar products as observed in the one-pot assembly.

However, for HE7-DNA and longer HEn-DNA, nonpenetrating materials along with unbound cubes were

mostly obtained. This could indicate that the assemblies generated from the stepwise protocol could be

kinetic products.

Supplementary Figure 58. Comparison of stepwise assembly of preformed C4 with annealed HEn-DNA

and one-pot assembly of C4 with HEn-DNA (5% native PAGE).

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Due to the possibility of following the formation of their higher-order products on native PAGE,

C4/HE6-8-DNA was chosen to investigate in more details on the effect of HEn-DNA concentration and the

incubation temperature. C4 was assembled in 1xTAMg at 250 nM. Separately, HEn-DNA was annealed at

two concentrations (1.5 and 5 µM) in 1xTAMg. To 5 µL of C4 was added either 5 µL of 1.5 µM HEn-DNA

or 1.5 µL of 5 µM HEn-DNA plus 3.5 µL of 1xTAMg to obtain final C4 and HEn-DNA concentrations of

125 nM and 750 nM, respectively. The mixtures were incubated for 30 minutes either at room temperature

or at 37oC in the water bath, and then analyzed by native PAGE. Parts of the results (preannealed HEn-DNA

at 5 µM) were discussed in the article. In Supplementary Fig. 59, for the incubation at room temperature,

all HEn-DNA tended to form larger higher-order structures with C4 as indicated by the smearing and the

presence of nonpenetrating materials, and the presence of unbound cube C4 suggests that the binding of C4

to HEn-DNA was not efficient at room temperature. For the incubation at 37oC, the smaller higher-order

structures, such as a cube dimer, formed better and the bands were cleaner, particularly for C4/HE8-DNA.

Supplementary Figure 59. Stepwise assembly of preformed C4 with annealed HE6-8-DNA (5% native)

PAGE. For stepwise assembly, low and high annealed concentrations of HEn-DNA were 1.5 and 5 µM,

respectively.

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C4/HE8-DNA is likely to require higher incubation temperature to form the smaller higher-order

structures such as cube dimer and trimer as observed in one-pot assembly. To verify this, C4 was assembled

in 1xTAMg at 250 nM. Separately, HE8-DNA was annealed at both 1.5 and 5 µM in 1xTAMg. To 5 µL of

C4 was added either 5 µL of 1.5 µM HE8-DNA or 1.5 µL of 5 µM HE8-DNA plus 3.5 µL of 1xTAMg to

obtain final C4 and HE8-DNA concentrations of 125 nM and 750 nM, respectively. The mixtures were

incubated at various temperatures for 30 minutes on the thermocycler then analyzed by native PAGE. In

Supplementary Fig. 60, the formation of the smaller higher-order structures was better at higher incubation

temperatures, most likely to be optimized in the range of 45-50oC. However, thermal denaturation of C4

(Tm of C4/DNA = 54.6±1.8oC) and unbinding of HE8-DNA from C4 also occur at higher temperature as

well.

Supplementary Figure 60. Stepwise assembly of preformed C4 with annealed HE8-DNA (5% native

PAGE). For stepwise assembly, low and high annealed concentrations of HEn-DNA were 1.5 and 5 µM,

respectively. The incubation temperatures were room temperature, 37.0oC, 41.3 oC, 45.9 oC, 51.0 oC and

55.1 oC.

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Similarly, the stepwise assembly of C8 and HE6-DNA was carried out. C8 and HE6-DNA were

separately annealed in 1xTAMg at 250 nM and 5 µM, respectively. To 5 µL of C8 was added 3 µL of 5 µM

HE6-DNA and 2 µL 1xTAMg to obtain final C8 and HEn-DNA concentrations of 125 nM and 1.5 µM,

respectively. The mixtures were incubated for 30 minutes either at room temperature or at 37oC in water

bath, and then analyzed by native PAGE. In Supplementary Fig. 61, there was no difference between the

products obtained from the one-pot assembly and the stepwise assembly. Interestingly in the stepwise

assembly, HE6-DNA micelles can bind and fit themselves in the interior of C8 scaffolds which are slightly

smaller than the micelles. This generated only one product (a cube with hydrophobic core) without cross-

linking the cubes that will lead to higher-order structures. Therefore, it is very likely that HE6 chains are

very dynamic such that the hydrophobic core of the HE6-DNA micelle is not in “frozen” state.

Supplementary Figure 61. Stepwise assembly of preformed C8 with annealed HE6-DNA (5% native

PAGE).

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XVIII. Effect of the cage geometry on the assembly of higher-order structures

The concentration of each cage was kept constant at 125 nM while HE6-DNA was added in

appropriate 1.5x stoichiometric concentrations (such as 750 nM for C4). For one-pot assembly, all required

strands were annealed together in 1xTAMg (10 µL), and then analyzed on native PAGE. In Supplementary

Fig. 62, an assembly of combined two different cages showed two bands corresponding to individual cage

(lane 4-6), indicating that each cage can efficiently assemble in the mixtures of the two cages. With the

addition of HE6-DNA, all possible structural combinations were observed (for example, in the combination

of TP3 and C4 with HE6-DNA, there were homodimers of TP3/TP3 and C4/C4 and heterodimer of TP3/C4).

Similar combination was also observed for other higher-order structures of TP3 and C4 as well as in other

cage combinations (TP3/PP5 and C4/PP5).

Supplementary Figure 62. Combination of cages and their assembly with HE6-DNA (5% native PAGE).

Cage can form efficiently in the mixture of clip components for two cages. Addition of HE6-DNA generated

both homocages (i.e., dimer of TP3 and TP3) and heterocages (i.e., dimer of TP3 and C4).

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Next, the stepwise assembly was performed to investigate the stability of preformed higher-order

structures of each cage in the mixture of the two types. Each cage at 125 nM was annealed with HE6-DNA

in 1xTAMg (10 µL). Then, 5 µL of one mixture was mixed with 5 µL of another mixture (i.e., TP3/HE6-

DNA and C4/HE6-DNA). The combined mixture was incubated for 30 minutes at either room temperature

or 37oC before analyzing on native PAGE. In Supplementary Fig. 63, no visible heterodimer was observed

at room temperature incubation (lane 7-9), indicating the good stability of the preformed assembly at room

temperature. However, the fainted bands of heterodimers can be seen at 37oC incubation (lane 10-12).

Supplementary Figure 63. Combination of cages and their assembly with HE6-DNA (5% native PAGE).

In the one-pot assembly, both homodimers and heterodimers were observed. In case of the stepwise

assembly, the homodimers were stable at room temperature incubation and no exchange between two

homodimer population was observed.

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To further support the existence of heterocages, the fluorescent-labelling strategy was employed to

differentiate between two types of cages: trigonal prism TP3,AC (1AB, 2AC, TP3-AB) was labelled with

Cy5-C (Cy5-TTTTTTCTTACGGCAGAGT) and cube C4,AD (1AB, 2AB, 3AD, 4AB) was labelled with D-

Alexa488 (ATGGACCAAGGCCATTTTT-Alexa488). The co-localization of these two dyes can indicate

the heterocage structures. In Supplementary Fig. 64, the stepwise assembly of TP3,AC-Cy5/HE6-DNA and

C4,AD-Alexa488/HE6-DNA at 37oC for 30 mins leaded to the heterodimer formation as indicated by faint

orange band between red and green dimer bands (right gel). Although the intensity of this heterodimer band

was low, GelRed staining (left gel) can further support the presence of this band.

Supplementary Figure 64. Combination of cages and their assembly with HE6-DNA (5% native PAGE).

TP3 and C4 were tagged with fluorescent dyes Cy5 (red) and Alexa 488 (green), respectively. The co-

localization of these two dyes can indicate the formation of heterocage between TP3 and C4.

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XIX. Isolation of the higher-order structures of C4/HE6-DNA and C4/HE7-DNA

C4 (125 nM) and HE6-DNA/HE7-DNA (750 nM) were annealed in 1xTAMg. Then, 100 µL of

sample was mixed with 20 µL of glycerol mix. On 5% native PAGE, 12 µL of the mixture was loaded as a

reference in one lane while the remaining 108 µL was loaded in other lanes for the isolation process. The

gel was run at 250 V for 2.5 hours with 1xTAMg as the running buffer. The part of the gel containing

reference band was cut and stained with GelRed in order to measure the distances of the target bands from

the well (analogous to Rf value). The bands on another part of the gel was then excised according to the

calculated distances, cut into small pieces and soaked in 1xTAMg for 1-2 days at 4oC. After that the isolated

samples were analyzed on 5% native PAGE. In Supplementary Fig. 65, all isolated higher-order bands were

stable and survived the isolation process. The smallest higher-order assembly (cube dimer) did not seem to

re-equilibrate to form the mixture of the higher-order structures. However, for the larger higher-order

structures (cube trimer and tetramer), there might be some or little re-equilibration.

Supplementary Figure 65. Isolation of the assembled products of C4 with HE6-DNA/HE7-DNA.

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DLS measurements were carried out for each of the isolated C4/HE6-DNA higher-order structures

(Supplementary Fig. 66). The samples were filtered with 0.22 µm syringe filter to remove any particle that

could interfere with the measurement. The results are summarized in Supplementary Table 14. Both

structures showed similar ranges of sizes. AFM was performed in order to visually characterize the

morphology of the isolated products; however, no structure was observed (data not shown).

Supplementary Table 14. Hydrodynamic radii and % polydispersity of isolated C4/HE6-DNA obtained

from DLS

Structure Rh (nm) % polydispersity

Dimer (lower) band 7.2±0.7 39.4±13.9

Trimer (upper) band 8.0±0.7 42.3±3.1

Supplementary Figure 66. DLS results of C4/HE6-DNA isolated by PAGE. Left: histogram showing size

distribution of the assemblies; right: its intensity correlation function.

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XX. Effect of concentration on the stability of the assemblies

C4 with HE6-8-DNA was prepared at two concentrations (125 nM C4 and 1250 nM with respect to

C4) in 1xTAMg and then subjected to thermal annealing protocol as described in section V. For 5% PAGE

analysis, 10 µL of 125 nM C4/HE6-8-DNA and 10 µL of 10x diluted 1250 nM C4/HE6-8-DNA were mixed

with 2 µL glycerol mix and loaded on the gel. In Supplementary Fig. 67, the assembly at high concentration

(1250 nM C4) still resulted in the expected higher-order structures for C4/HE6-DNA and C4/HE7-DNA but

the products were not as clean as those assembled directly at lower concentration (125 nM C4). In case of

HE8-DNA, only larger higher-order structures were observed, suggesting that the assembly is

concentration-dependent.

Supplementary Figure 67. Concentration-dependent assembly of C4/HE6-8-DNA (5% native PAGE). The

1x and 10x assembly concentration were 125 nM and 1250 nM with respect to the cube C4, respectively.

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It is also interesting to determine the stability of the assembly upon the dilution. C4/HE6-DNA was

assembled at 125 nM C4 in 1xTAMg. Then, it was diluted with 1xTAMg to different concentrations before

analyzing on 5% native PAGE. The structures in Supplementary Fig. 68 were labeled with Cy3-B (Cy3-

TTTTTCCATCTGGTATTAC). The gel was imaged under Cy3 emission. The result indicates that the

assembly was stable up to 40x dilution (~3 nM).

Supplementary Figure 68. Dilution of C4/HE6-DNA after the assembly. 5% native PAGE.

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XXI. References

(1) Edwardson, T. G. W.; Lau, K. L.; Bousmail, D.; Serpell, C. J.; Sleiman, H. F. Nat. Chem. 2016, 8,

162.

(2) Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G. Anal. Chem. 2007, 79, 4215.

(3) Serpell, C. J.; Edwardson, T. G.; Chidchob, P.; Carneiro, K. M.; Sleiman, H. F. J. Am. Chem. Soc.

2014, 136, 15767.

(4) Edwardson, T. G.; Carneiro, K. M.; Serpell, C. J.; Sleiman, H. F. Angew. Chem., Int. Ed. 2014, 53,

4567.

(5) Eryazici, I.; Prytkova, T. R.; Schatz, G. C.; Nguyen, S. T. J. Am. Chem. Soc. 2010, 132, 17068.