SUPPLEMENTARY INFORMATION - .1Department of Chemistry and The Skaggs Institute for Chemical Biology,

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  • SUPPLEMENTARY INFORMATIONdoi: 10.1038/nmat2877

    nature materials | www.nature.com/naturematerials 1

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    Supplementary Information

    DNA-controlled assembly of an NaTl lattice structure from gold and protein nanoparticles

    Petr Cigler,1, Abigail K. R. Lytton-Jean2,, Daniel G. Anderson2,3,

    M.G. Finn1,*, and Sung Yong Park4,* 1Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research

    Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA 2David H. Koch Institute for Integrative Cancer Research; 3Department of Chemical

    Engineering and Harvard-MIT Division of Health Sciences & Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA

    4Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, 601 Elmwood Ave., NY 14642, USA

    These authors contributed equally to this work

    Chemical syntheses of Q-DNA conjugate Chemicals were obtained from Fisher Scientific or Sigma-Aldrich. All solvents were dried

    by passage through activated alumina columns, unless otherwise specified. TLC was performed on silica (Merck). Compound 1 was prepared as described by Chang and coworkerss1. Aminohexylated, HPLC grade DNA (CG-3), was obtained from IDT (USA). THPTA was prepared according the procedure described by Finn and coworkers27. Q capsids were prepared and decorated by azide-terminated linkers as described previously25,26,s2.

    A. Fluorescent heterobifunctional BODIPY-based spacer

    NNB

    Cl

    FF

    a)

    b) NNB

    HN

    FF

    c)

    NNB

    N

    FF

    O CO2H

    1 2 3 Figure S1. Synthesis of BODIPY-linker. a) KI, acetone; b) propargylamine; c) succinyl anhydride, DMAP, pyridine.

    1) Synthesis of 8-propargylaminomethyl-2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (compound 2 in Figure S1) Compound 1 (1.00 g, 2.84 mmol) was dissolved in 150 mL of dry acetonitrile under argon.

    Potassium iodide (1.18 g, 7.09 mmol, 2.5 eq) was added and the reaction mixture was refluxed for 15 min. After cooling to room temperature, propargylamine (1.56 g, 28.4 mmol, 10 equiv)

    Supplementary InformatIon for:

    Dna-controlleD aSSembly of a natl lattIce Structure from golD nanopartIcleS anD proteIn nanopartIcleS

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    was added and the solution was refluxed until the violet spot of the iodo derivative disappeared from the TLC (ca. 30 min.).

    The solvents were then removed under vacuum to dryness. The solids were dissolved in dichloromethane and extracted twice by water. The organic layer was dried with anhydrous MgSO4 and evaporated under vacuum. The yield was practically quantitative and the crude product was used directly for the next reaction step.

    MS (ESI): [M F]+ 353.2, [MH]+ 373.2, [MNa]+ 394.1; TLC (dichlomethane): Rf = 0.17 (bright, orange fluorescent spot). 2) Synthesis of Compound 3

    Compound 2 obtained from previous reaction was dissolved in 10 mL of dry dichloromethane. Succinic anhydride (1.42 g, 14.1 mmol), a catalytic amount of 4-(dimethylamino)pyridine (69 mg, 0.57 mmol), and dry pyridine (4.2 mL) were added and the reaction mixture was stirred at room temperature overnight. The solvents were evaporated under vacuum to dryness. The crude product was chromatographed on silica using gradient (2 10% of MeOH in dichloromethane).

    Yield: 0.44 g (33 %); TLC (dichlomethane): Rf = 0.12 (bright, orange fluorescent spot). 1H NMR (CDCl3): 1.00 (t, 6H), 2.15 (s, 1H), 2.18 (s, 6H), 2.35 (q, 2H), 2.48 (s, 6H), 2.96 (t, 2H), 3.06 (t, 2H), 3.98 (s, 2H), 4.89 (s, 2H). MS (ESI): [M F]+ 452.2, [MNa]+ 494.2; Fluorescence spectra (in methanol): (excitation) = 540 nm, (emission) = 553 nm.

    B. Preparation of Q-DNA conjugates 1) Synthesis of BODIPY-DNA conjugates

    Figure S2. Synthesis of BODIPY-DNA conjugate.

    EDCHCl

    H2NO A10 AAGACGAATATTTAACAA

    5' 3'3 +

    NNB

    N

    FF

    O

    CG-3

    NH

    OO A10 AAGACGAATATTTAACAA

    5' 3'

    NEt3N

    O

    O

    HO

    SO3NaDMSO/aq base

    BoDIPY-DNA conjugate

    Compound 3 (25 mg, 52 mol) was dissolved in dry DMSO (327 L). EDC hydrochloride (23.8 mg, 125 mol) and triethylamine (12.5 mg, 125 mol) were dissolved in dry DMSO (123 L). The solutions were combined and gently mixed for 15 min. The solution was then diluted to 3.0 mL with dry DMSO and N-hydroxysulfosuccinimide (67 mg, 312 mol) was added. After 15 min. of gentle mixing, an ice-cold solution of CG-3 (11 mg, 1.25 mol) in 2900 L of 0.25 M carbonate buffer (pH = 8.5) was added in small parts while cooling the reaction tube with ice.

    After addition of all DNA, the reaction mixture was incubated for 15 min, after which a new portion of EDC hydrochloride (23.8 mg, 125 mol) and 150 L of 3.3% solution of NaOH (125 mol) were added. At this point, the solution became slightly turbid. After 3.5 h, the reaction mixture was diluted with 54 mL of water and centrifuged at 3500 rpm. The supernatant

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    was lyophilized, dissolved in 20 mL of water and purified by gel permation chromatography on NAP-25 columns. The combined fractions of eluate were diluted to 35 mL. Acetate buffer (3 M, pH = 5.3, 3.5 mL) and absolute ethanol (105 mL) were added and the solution was chilled at 80 C for 4 h. The resulting suspension was centrifuged (14 000 rpm, 30 min) at low temperature, the supernatant removed and the precipitated solid was rapidly and gently washed with 1 mL of 80 C absolute ethanol.

    The conjugate (Figure S2) was dissolved in water to approx. 1.5 mM solution and its specific concentration was determined by UV spectrophotometry. The purity of the conjugate was checked by HPLC and the stock solution was stored at 4C. MALDI-TOF mass spectrometry: (M + H+) calculated, 9288; found 9289. 2) Synthesis of Q-DNA conjugates

    The icosahedral Q virus-like protein nanoparticle (VLP) is approximately 28 nm in diameter and is composed of 180 identical proteins, each of which displays four amino groups accessible on the outer surface25,26,s2. The surface amino groups were acylated using an excess of azide-containing N-hydroxysuccinimide linker 1 (Figure 3c in the main text). The BODIPY-DNA-alkyne conjugate described above was then coupled to the particle-displayed azide groups using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reactions3.

    Degassed aqueous solutions of reagents were sequentially mixed at room temperature according to Table S1. The reaction mixture was bubbled with nitrogen for at least one minute between each addition. After the last reagent was added, the Falcon tube was closed, sealed by Parafilm, and gently agitated for 8 h. The resulting crude conjugate was purified on a Sepharose 6000 GPC column and concentrated by membrane centrifugation. The degree of derivatization of the particles was estimated using UV-vis spectroscopy and ICP AES (quantifying boron from the BODIPY dye). Both techniques gave values of 19020 DNA chains per VLP, representing a more densely arrayed set of oligonucleotide connectors on the icosahedral surface than we achieved in our earlier work, which relied on standard lysine acylation or thiol alkylation chemistry24.

    Reagent Stock conc. Volume [L] Final conc. THPTA 50 mM 71.5 2.0 mM

    Sodium ascorbate 500 mM 22.7 6.3 mM CuSO4 50 mM 22.7 0.63 mM

    HEPES buffer (pH = 7.9) 0.1 M 653 36 mM NaCl 1 M 450 0.25 M

    BODIPY-DNA conjugate 1.3 mM 343 0.25 mM Azide groups on Q-azide 0.98 mM 228 0.125 mM

    Table S1. List of Regents for Synthesis of Q-DNA conjugates

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    Synthesis of DNA-linked nanoparticle crystals 10 -30 nm gold nanoparticles (AuNPs) modified with synthetic oligonucleotides (5HS-

    A10-AAG ACG AAT ATT TAA CAA 3) were prepared according to literature proceduress4. Linker-A is complementary to the DNA immobilized on the Q VLP surface (region 1)

    and has a dangling end composed of four bases (5CGCG 3) (region 2, Figure S5a). To direct the assembly of Q VLPs into a close-packed FCC structure, DNA linker-A was added to the Q VLPs above the melting temperature of region 1 followed by slow cooling to room temperature (10 min/1 C), Figure S5a. Alternatively, by simulating a binary system, the Q VLPs and AuNPs were assembled into a non-close packed NaTl structure. This was achieved by utilizing different linker sequences and hybridization temperatures. When linker-X was employed, containing a non-self-complementary dangling end (5 AAAGGAA 3), a second linker-Y with a complementary dangling end (5TTCCTTT 3), was required to achieve particle assembly, Figures 1c and S6. Therefore, because AuNPs hybridized to linker-X (AuNP-X) can only bind to Q VLPs hybridized to linker-Y (Q-VLP-Y), a two component system could be simulated. The interconnecting DNA can be described as containing two regions, where region 1 melts at ~55 C and region 2 melts at ~40 C. Therefore, region 2 can be thermally addressed while region 1 remains intact. This allowed us to create a binary system where AuNPs have Linker-X and Q VLPs have Link-Y hybridized to the surface. In an analogous fashion, decreasing the temperature of the system effectively increases the attractive force between the nanoparticles, inducing the formation of a different crystal phase. Therefore, by employing the correct choice of DNA linkers and hybridization temperature, programmable colloidal crystallization can be achieved without chemically alteri