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Accessibility of Densely Localized DNA on Soft Polymer Nanoparticles Munira F. Fouz,†,‡,∥ Sourav K. Dey,†,‡,⊥ Kosuke Mukumoto,†,§ Krzysztof Matyjaszewski,†,§ Bruce A. Armitage,*,†,‡ and Subha R. Das*,†,‡ †
Department of Chemistry, ‡Center for Nucleic Acids Science and Technology, and §Center for Macromolecular Engineering, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States
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S Supporting Information *
ABSTRACT: The dense localization of DNA on soluble nanoparticles can lead to effects distinct from equivalent amounts of the DNA in solution. However, the specific effect may depend on the nature of the assembly and the nanoparticle core. Here we examine the accessibility of densely packed DNA duplexes that extend from a bottlebrush polymer core. We find that unlike spherical nucleic acids, the DNA duplex bristles on the bottle-brush polymer remain accessible to sequence-specific cleavage by endonucleases. In addition, the hybridized strand of the duplex can be displaced through a toehold-mediated strand exchange even at the polymer interface. These results demonstrate that the DNA on bottle-brush polymer remains sufficiently flexible to allow enzymatic degradation or DNA hybridization.
1. INTRODUCTION Nanoparticles have proven to be useful scaffolds to localize small molecules and fluorescent labels for various biological applications. The compact covalent confinement of DNA on core materials offers, among other advantages, the ability to hybridize a specific complementary strand through the collective interactions of tens to hundreds of individual, densely packed DNA duplexes.1 The literature abounds in reports of the profound changes to the thermodynamics of hybridization within dense DNA shells leading to elevated thermal stability,2 sharper melting transitions, and enhanced binding constants relative to those of isolated duplexes free in solution.3 Such compact nucleic acid assemblies have the potential to transform individually weak hybridization interactions into substantially stable collective interactions that can enhance the overall functions of the nanoparticle.4−6 Therefore, the properties of DNA densely assembled on nanoparticles are of significant interest. A prominent example of dense DNA assemblies in a composite material is from Mirkin and co-workers: colloidal gold nanoparticles associate with oligonucleotides that contain a 3′- or 5′-terminal alkyl-thiol to form a spherical nucleic acid.7,8 The terminal thiol acts as a robust anchoring group on a specific terminus of the DNA strands to serve the nontrivial role of orienting all of the oligonucleotides in a common surface-normal direction.9 This control over the directionality ensures the availability for hybridization to their complements.10 High ionic strength allows prepared Au nanoparticles to be densely coated by thiol-functionalized DNA.11−13 The tight packing of DNA and corresponding high local salt concentrations in these nanostructures are reflected in their resistance to nuclease digestion.14 © XXXX American Chemical Society
An alternative approach to confining oligonucleotides in a dense assembly combines synthetic polymers with nucleic acids. The nucleic acid and polymeric components are considered to be blocks which can be conjugated in aqueous solution or while the DNA is immobilized on a solid support to generate DNA-block copolymers (DBCs).15−17 Among these DBCs, amphiphilic analogues form micellar or spherical aggregates exhibiting a hydrophobic polymer core and an outer shell of ssDNA. In addition to the dense array of DNA that imparts new properties to the overall particle, the shape of the core material can also play an important role in applications such as cellular delivery and the labeling of targets. Rodlike particles composed of some DBC materials have shown an order of magnitude more efficient cellular internalization than their spherical counterparts.18,19 We have recently described a polymer core that has a bottlebrush architecture and is functionalized with hundreds of DNA duplexes attached to the tips of the bottle-brush “bristles”.20 On this soft nanoparticle, the DNA bristles can act as a dense scaffold for fluorescent intercalating dyes. Significantly, we found that the dense DNA scaffold prevented the dissociation of the intercalating dyes under conditions where the same dyes would rapidly dissociate from isolated DNA nanostructure scaffolds, allowing the assembly of antibody-based “nanotags” with extremely high extinction coefficients for fluorescent labeling applications.20 Special Issue: Nucleic Acids Nanoscience at Interfaces Received: June 15, 2018 Revised: August 6, 2018
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DOI: 10.1021/acs.langmuir.8b02038 Langmuir XXXX, XXX, XXX−XXX
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Figure 1. DNA bottle-brush polymer. A bottle-brush polymer provides a scaffold for densely arrayed DNA conjugated by click chemistry and the annealing of a second strand that includes a fluorescent Cy5 label. Nearly 200 strands of duplex DNA are on each polymer nanoparticle, for which a more compact representation is used to show the reaction. 50 kDa molecular weight cutoff filters. This filtration step allows the large BBPs to be retained in the membrane filter while cleaved DNA passes though the membrane. Because the cleaved DNA includes 5′terminal Cy5 or Cy3, the extent of the reaction can be monitored with respect to the fluorescence intensity of the filtrate. After the filtrate was collected, the retained BBPs were collected by reverse spinning the filters. The residual fluorescence from uncleaved DNA was measured in parallel to the filtrate. Fluorescence intensities were measured at λex = 615 nm for Cy5 and at λex = 515 nm for Cy3. 2.4. Strand Displacement Experiments. The complementary DNA strands (A/Cy5-Acomp) were annealed in phosphate-buffered saline (PBS; pH 7.5, Na+ = 100 mM) to obtain a final concentration of 100 nM duplex DNA on the DBBPs or in free solution. The duplex DNA was saturated with YOYO-1 (500 nM, purchased from Life Technologies) prior to displacement reactions. A/Cy5-Acomp duplexes (200 μL) were excited at 450 nm to get the initial fluorescence signal. The slit width for both excitation and emission monochromators was 5 nm. The A′ strand and the AC′ strand, which are fully complementary to the A and AC strands, respectively, were added (to give 100 nM, 500 nM, and 2 μM final concentrations) to the preannealed 100 nM A/Cy5-Acomp duplex-containing solutions and mixed vigorously, and the fluorescence signal was measured immediately by exciting at 450 nm. The fluorescence emission was also recorded at 1 min, 6 min, 10 min, 30 min, 1 h, 2 h, and 15 h time points. The duplexes for the reduced DNA density sample were prepared by annealing Cy5-Acomp strands to DNA on A and PEO clicked DBBPs to give a 40 nM duplex concentration in a 200 μL volume of PBS buffer (pH 7.5, Na+ = 100 mM). The AC′ strand was added to give a 200 nM final concentration, and after the solution was mixed, and the fluorescence was measured immediately. The fluorescence emission was also recorded at 1 min, 6 min, 10 min, 30 min, 1 h, 2 h, and 15 h time points. The samples were stored under dark conditions throughout the process.
Here we explore the accessibility of this dense array of DNA on the bottle-brush polymer (BBP).20 Unlike a more rigid scaffold such as a gold nanoparticle, the BBP has a flexible backbone based on a methacrylate polymer which might permit large nuclease enzymes or DNA oligonucleotides to gain access to the DNA matrix. Here we demonstrate the ability of endonuclease enzymes to cleave DNA-conjugated BBPs (i.e., DBBPs) at internal sites as well as the ability of other DNA strands that use a toehold sequence to mediate the displacement of either strand in a DBBP. The flexibility of the polymeric BBP core likely provides variability to the local density of the DNA that permits accessibility to the DNA.
2. MATERIALS AND METHODS 2.1. Oligonucleotides. DNA sequences with 5′-terminal hexynyl and 5′-Cy5 and 5′-Cy3 modifications were synthesized using solidphase oligonucleotide synthesis on a MerMade-4 synthesizer (Bioautomation, Plano, TX, USA). Commercially available starting materials were used without further purification. Phosphoramidites (dA, dC, dG and T) for DNA synthesis with labile PAC protecting groups and appropriate reagents were purchased from Chem Genes (Wilmington, MA, USA) and Glen Research (Sterling, VA, USA). Synthesis and deprotection of the oligonucleotides were conducted under standard protocols for PAC-protected amidites, as recommended by the manufacturers. The DNA synthesis columns were purchased from Biosearch Technologies, Inc. (Novato, CA, USA). Any unmodified DNA oligonucleotides were purchased from Integrated DNA Technologies, Inc. and obtained as lyophilized powders. 2.2. DBBP Synthesis. The synthesis of DBBPs containing (a) 100% DNA or (b) 40% DNA and 60% poly(ethylene oxide) (PEO) clicked onto the bristles was described previously.20 The polymer backbone was composed of poly[2-(2-bromoisobutyryloxy)ethyl methacrylate] ca. 400 units long, from which about 200 side chains of 2-(2-(2-methoxyethoxy)ethoxy)ethyl methacrylate (MEO3MA) were grafted via the terminal bromide.21,22 Replacing the terminal bromides with azide groups provided the means to covalently attach a 5′-hexynyl-modified DNA strand through the copper-catalyzed azide alkyne cycloaddition reaction.23,24 2.3. Endonuclease Activity on DBBPs. dsDNA (1 μM) on DBBPs with A/Cy5-Acomp and/or B/Cy3-Bcomp strands (with restriction sites for BamHI and EcoRI, respectively) was prepared in phosphate-buffered saline (PBS; pH 7.5, Na+ = 100 mM). A buffer exchange procedure was followed with 50 kDa molecular weight cutoff filters. A/Cy5-Acomp containing BBPs was buffer exchanged to final buffer conditions of 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, and 100 μg/mL BSA at pH 7.9 using NEBuffer BamHI (purchased from New England Biolabs Inc.) The same buffer exchange procedure was followed for B/Cy3-Bcomp containing BBPs to bring the final buffer conditions to 100 mM NaCl, 50 mM TrisHCl, 10 mM MgCl2, and 0.025% Triton X-100 at pH 7.5 using NEBuffer EcoR I purchased from New England Biolabs Inc. Doublestranded DNA (∼1 μg total in 500 μL) in respective buffers was treated with 10, 20, and 50 units of BamHI and EcoRI enzymes separately and was mixed and incubated in a 37 °C water bath for 1 h. At the end of the incubation period, the solutions were filtered using
3. RESULTS AND DISCUSSION We previously described a bottle-brush polymer (BBP)20 via the atom-transfer radical polymerization (ATRP) method25−27 that used a poly[2-(2-bromoisobutyryloxy)ethyl methacrylate] (PBiBEM) macroinitiator with a degree of polymerization of 400, from which about 200 side chains of 2-(2-(2-methoxyethoxy)ethoxy)ethyl methacrylate (MEO3MA) are grafted.21,22 At the nearly 200 side-chain ends, the installation of an azide provides the means to covalently attach hundreds of copies of an alkyne-modified DNA oligomer (23-mer) through the copper-catalyzed azide alkyne cycloaddition (CuAAC or click) reaction.20 This DNA-BBP (DBBP) with single-stranded DNA (ssDNA) oligomer bristles could then be transformed to a DBBP with double-stranded DNA (dsDNA) by simply hybridizing a complementary strand (Figure 1; see also Supporting Information Scheme S1A for chemical structures and Supporting Information Table S1 for DNA sequences). The approximately 200 dsDNA bristles on the DBBP could be loaded with bis-intercalating fluorescent dye YOYO-1 (ca. 5 YOYO-1 molecules per DNA bristle). When the hybridized DNA strand bore a terminal Cy5 fluorescent acceptor dye, B
DOI: 10.1021/acs.langmuir.8b02038 Langmuir XXXX, XXX, XXX−XXX
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Figure 2. Sequence-specific restriction endonuclease cleavage of the DNA on bottle-brush polymers (DBBPs). (A) A/Cy5-Acomp duplexes with the palindromic 5′-GGATCC stretch are accessible to the BamHI enzyme (but not EcoRI). (B) The Cy5 signal (λex = 615 nm) on the DBBPs decreases with increasing BamHI concentration (left spectra) while the signal increases correspondingly in the filtrate (right spectra). (C) B/Cy3Bcomp duplexes with the 5′-GAATTC sequence are susceptible to EcoRI (but not BamHI). (D) With increasing addition of EcoRI, the Cy3 signal (λex = 515 nm) on the DBBPs decreases (left spectra) while the signal increases in the filtrate (right spectra).
energy transfer27−29 from the intercalated YOYO-1 resulted in greatly enhanced fluorescence emission of Cy5.20 Additionally, because of the highly dense localization of dsDNA on the brush, intercalated YOYO-1 molecules did not dissociate from the DBBP to competing DNA (nonbrush dsDNA that was added to the solution). That this was dependent on the high density of DNA on the DBBP could be verified as DBBPs with a lower loading of dsDNA (40% compared to the original DBBP, with the remaining 60% of the bristles functionalized with a poly(ethylene oxide) chain), did not retain the YOYO-1 dyes. This high retention of the dyes on the DBBPs was useful in applying the DBBPs as antibody conjugates (i.e., nanotags) for fluorescence imaging and detection applications.20 Here we examine if the high-density localization of the DNA on the flexible DBBP imparts other properties to the DNA that would not be available to an equivalent amount of (nonbrush) DNA in solution. Mirkin and co-workers have shown that the high density of DNA localized on a gold nanoparticle in spherical nucleic acids (SNAs) hinders nuclease degradation.14 Other recent reports describe the nuclease cleavage of RNA sites closer to the particle core on SNA assemblies, despite their seemingly dense structure.30,31 To assess the nuclease sensitivity of the DNA strands assembled on the DBBP, we treated the DBBPs with restriction endonucleases BamHI and EcoRI (Figure 2) and prepared DBBPs with the corresponding recognition sequences for these two enzymes. We chose endonucleases that would target internal sites within the DNA strands to assess their accessibility and included a 5′-terminal fluorescent dye on the DNA strands as reporters. The terminal fluorescent dye is not expected to hamper the activity of endonucleases that cleave DNA at internal positions unless the high density of strands and the associated local environment are factors. We first tested the effect of BamHI, a restriction endonuclease that is unique in that it undergoes a series of
unconventional conformational changes upon DNA recognition.32 This allows the DNA to maintain its normal B-DNA conformation without distorting to facilitate enzyme binding.33 We added BamHI in manufacturer-recommended, 1-, 2-, and 5-fold excesses to the DBBP with dsDNA based on A (red strand, Figure 2A) and Cy5-Acomp (blue strand with blue sphere) and incubated at 37 °C for 1 h with frequent mixing of the solution. We find that BamHI cleaves the dsDNA on the DBBP having the cognate palindromic recognition sequence 5′-GGATCC-3' (Figure 2A). The cleavage can be monitored by the fluorescence of the Cy5-DNA that is either retained or passes through a membrane filter. The initial fluorescence signal from uncleaved DNA (in the absence of BamHI, Figure 2B, blue spectrum) decreases with the BamHI concentration, with a 5-fold excess giving nearly complete cleavage (Figure 2B, left). In good agreement with the decreasing fluorescence intensity of Cy5 on the DBBPs, the Cy5 signal in the filtrate increased with increasing BamHI concentration (Figure 2B, right). Although an excess of the enzyme (relative to the concentration recommended by the vendor to cleave a similar amount of nonbrush DNA under similar buffered conditions) is required, the densely localized DNA on the DBBPs still remains accessible to and is efficiently cleaved by BamHI. To confirm not just the accessibility of the dense DNA on DBBPs to enzymatic cleavage but also the sequence specificity of the enzyme activity on these DNAs, we used DBBPs with click conjugated DNA sequence B and hybridized the complementary Cy3-labeled Bcomp strand. These DBBPs have dsDNA bristles with the recognition sequence of the EcoRI enzyme (5′-GAATTC-3′, Figure 2C). EcoR I was added to the reaction mixtures in 1- and 2-fold excesses relative to the recommended amount and incubated at 37 °C for 1 h with frequent mixing of the solution. After 1 h, the reaction mixture was filtered through 50 kDa molecular weight cutoff filters. Similar to the previous procedure discussed for the BamHI C
DOI: 10.1021/acs.langmuir.8b02038 Langmuir XXXX, XXX, XXX−XXX
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Figure 3. Strand exchange of DNA on DBBPs. (A) The schematic shows 5 residue toehold 1 and toehold 2 of A (red strand)/Cy5-Acomp (blue strand) duplexes on the bottle-brush. A′comp (gray strand) displaces the Cy5-Acomp via toehold 1 and forms Cy5-Acomp/A′comp duplexes on the brush. Alternately, AC′comp (black) binds to toehold 2 and releases Cy5-Acomp/AC′comp duplexes and results in single-stranded Seq A. The same design is used on nonbrush DNA. The spectra show the fluorescence emission of YOYO-1 (λmax = 510 nm) and Cy5-sensitized emission ((λmax = 663 nm) when excited (λex = 450 nm). The sensitized emission of Cy5 (green spectra) decreases with the increased addition of (B) A′comp strand and (C) AC′comp strand. The disappearance of the Cy5 emission occurs with 20-fold excesses of A′comp strand (B, left spectra) and AC′comp (B, right spectra) for brush DNA while 10-fold excesses of A′comp strand (C, left spectra) and AC′comp (C, right spectra) are sufficient for the full displacement of strands for nonbrush DNA duplexes. The red spectra show the YOYO-1 fluorescence emission of A/Acomp duplexes. The starting DNA duplexes were saturated with YOYO-1 (500 nM) prior to displacement reactions.
reaction, the fluorescence of Cy3 was measured (λex = 515 nm). Figure 2D shows the decreasing fluorescence intensity of Cy3 on the DNA on the bottlebrush (left spectra) and the increasing fluorescence intensity of Cy3 in the filtrate (right spectra) with increasing EcoRI concentration. Neither DBBP
exhibited cleavage when incubated with the noncognate restriction enzyme, confirming the sequence-specificity of these enzymes to the DBBP-localized DNA. Following the test of accessibility of the DNA to enzymatic cleavage at internal positions, we tested the accessibility of the D
DOI: 10.1021/acs.langmuir.8b02038 Langmuir XXXX, XXX, XXX−XXX
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Figure 4. A high density of DBBP retains the assembled intercalated dyes. Fluorescence emission was recorded (λex = 450) at different time points after the addition of the AC′comp strand in 20-fold excess. (A) The sensitized emission from Cy5 does not reappear even after 15 h, suggesting that the high DNA density prevents the dissociation of positively charged YOYO-1 in Cy5-Acomp/AC′comp duplexes in solution (left spectra). In contrast, sensitized emission increases after 6 min of addition of AC′comp to the (B) nonbrush control sample and (C) DBBP with reduced DNA density (40% DNA and 60% PEO side chains).
reaction on an equivalent amount of nonbrush dsDNA and found qualitatively similar results (Figure 3B, right spectra). These data show that the displacement of a strand on the bottlebrush core is possible even when the toehold is located in the interior of the compact DNA shell of the bottlebrush. We repeated these displacement reactions with both brush and nonbrush duplexes using AC′comp that can bind to toehold 2 at the outer terminus away from the brush polymer. During the displacement, Cy5-Acomp/AC′comp duplexes are released into the solution, leaving behind single-stranded sequence A on the brush polymer. We observe a similar trend in displacement with decreasing sensitized emission of Cy5 for both brush (Figure 3C, left spectra) and nonbrush (Figure 3C, right spectra) systems. In principle, YOYO-1 molecules that were previously intercalated in A/Cy5-Acomp duplexes on the brush would be expected to dissociate from the single-stranded A on the brush in order to bind the double-stranded duplex in solution. If this were to happen, then energy transfer would be maintained, but this is clearly not observed. YOYO-1 added to brush or nonbrush DNA results in similar fluorescence emission with greater fluorescence from dsDNA than from ssDNA (Figure S1). Because all fluorescence measurements were recorded immediately after the mixing of the displacing strands, we monitored the sensitized emission signal at different time points to determine if YOYO-1 would dissociate from the single-stranded DNA on the brush. However, we did not observe any Cy5 signal due to sensitized emission even after 15 h (Figure 4A), indicating that YOYO-1 remains on the ssDNA of the DBBP. This suggests that even ssDNA due to the high local density can retain the charged fluorescent dyes. In contrast, nonbrush DNA showed a sensitized Cy5 signal from the YOYO-1 bound to Cy5-Acomp/AC′comp duplexes, within 1 min after the addition of AC′comp (Figure 4B), indicating rapid strand displacement and dissociation of YOYO-1 from the remaining ssDNA to bind to the Cy5-labeled dsDNA. To verify that the retention of YOYO-1 on the ssDNA on DBBPs was a result of the high local density of DNA, we repeated the same experiment with a DBBP scaffold with reduced DNA loading density (40% DNA and 60% PEO).20 In contrast to the DBBP with 100% DNA loading, the sensitized Cy5 emission appears within 1 min of mixing, similar to that of nonbrush DNA (Figure 4C), indicating facile dissociation of YOYO-1 after strand displacement from the lower-density DBBP.
DNA termini to strand displacement by a third DNA strand. A strand displacement reaction leads to the displacement of one or more prehybridized strands when two strands with partial or full complementarity hybridize to each other. The displacement reaction is initiated at complementary single-stranded domains (referred to as toeholds) and progresses through a random walk process.34 Various molecular devices, including circuits, catalytic amplifiers, autonomous molecular motors, and reconfigurable nanostructures, have been rationally designed to use DNA strand-displacement reactions.35 The progress of strand-displacement reactions is typically assayed using fluorescence via either reporter complexes that stoichiometrically react with the output or via dual-labeled probes as output strands.36 A strand-displacement reaction is central to the SNA-based “nanoflare” technology developed by Mirkin and co-workers for intracellular mRNA detection.37−39 We tested the accessibility of the DNA strands attached to bottlebrush scaffolds with a strand displacement scheme shown in Figure 3 using DBBPs that were loaded with intercalating YOYO-1 dyes. We used the DBBPs with conjugated 23-mer A (red strand) hybridized with Cy5-Acomp (blue strand) to give an 18 base pair duplex region flanked by 5 nucleotide toeholds proximal (toehold 1) and distal (toehold 2) to the polymeric core (Figure 3A). We tested two different displacing strands: A′comp (gray strand) and AC′comp (black strand) are 23-mer sequences that are fully complementary to either A or Cy5Acomp. A′comp can bind to toehold 1 and displace Cy5-Acomp, creating fully base paired (23-mer) duplexes on the bottlebrush and releasing single-stranded Cy5-Acomp into the solution, whereas AC′comp can bind to toehold 2, displacing as a duplex and leaving single-stranded A on the brush. To monitor the displacement reaction, we used the ability of the system to facilitate energy transfer. The acceptor dye (Cy5) shows sensitized emission when Cy5-Acomp is hybridized with A on the bottlebrush, and YOYO-1 is intercalated within the duplex to give a favorable proximity for energy transfer. When A′comp displaces Cy5-Acomp, the YOYO-1 dyes can no longer transfer energy to Cy5 (Figure 3A, top right); therefore, the resulting decrease in sensitized emission reports the extent of the displacement reaction. As shown in Figure 3B (left spectra), increasing amounts of A′comp lead to decreasing Cy5 emission and increasing YOYO-1 emission, indicating reduced energy transfer due to strand displacement, with the intercalators remaining bound to the duplex DNA on the brush. For comparison, we also monitored the displacement E
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We interpret the lack of energy transfer in the experiment with DBBPs with 100% DNA loading to indicate that strand displacement occurred, but the intercalator did not dissociate from the brush. In principle, the same result would be observed if the dye dissociated but strand displacement did not occur because the dye would no longer be associated with the Cy5labeled DBBP. However, the fact that we do not observe the dissociation of the dye from dsDNA-conjugated brushes (Figure 3B and our published work20) argues against this interpretation. Finally, we find it interesting that the fluorescence intensities of the samples consisting of YOYO-1 bound to ssDNA or dsDNA brushes are virtually identical, given the fact that YOYO-1 showed 2-fold-higher fluorescence for dsDNA versus ssDNA free in solution (Figure S1). This result indicates that the dense matrix of ssDNA on the brush offers binding sites that more effectively constrain the excited-state conformational mobility that directly impacts the fluorescence quantum yield of the dye compared to that of free ssDNA.40
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National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Bethesda, Maryland 20814, United States. ⊥ Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, United States. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS K.M. acknowledges NSF grant DMR 1501324, and B.A.A. and S.R.D. gratefully acknowledge financial support from the David Scaife Family Charitable Foundation (award 141RA01).
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(1) Kwak, M.; Herrmann, A. Nucleic Acid/Organic Polymer Hybrid Materials: Synthesis, Superstructures, and Applications. Angew. Chem., Int. Ed. 2010, 49 (46), 8574−8587. (2) Lytton-Jean, A. K. R.; Mirkin, C. A. A. Thermodynamic Investigation into the Binding Properties of DNA Functionalized Gold Nanoparticle Probes and Molecular Fluorophore Probes. J. Am. Chem. Soc. 2005, 127, 12754−12755. (3) Jin, R.; Wu, G.; Li, Z.; Mirkin, C. a.; Schatz, G. C. What Controls the Melting Properties of DNA-Linked Gold Nanoparticle Assemblies? J. Am. Chem. Soc. 2003, 125 (6), 1643−1654. (4) Zheng, J.; Zhu, G.; Li, Y.; Li, C.; You, M.; Chen, T.; Song, E.; Yang, R.; Tan, W. A. Spherical Nucleic Acid Platform Based on SelfAssembled DNA Biopolymer for High-Performance Cancer Therapy. ACS Nano 2013, 7 (8), 6545−6554. (5) Reed, A. N.; Putman, T.; Sullivan, C.; Jin, L. Application of a Nanoflare Probe Specific to a Latency Associated Transcript for Isolation of KHV Latently Infected Cells. Virus Res. 2015, 208, 129− 135. (6) Randeria, P. S.; Seeger, M. A.; Wang, X.-Q.; Wilson, H.; Shipp, D.; Mirkin, C. A.; Paller, A. S. siRNA-Based Spherical Nucleic Acids Reverse Impaired Wound Healing in Diabetic Mice by Ganglioside GM3 Synthase Knockdown. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (18), 5573−5578. (7) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 1996, 382 (6592), 607−609. (8) Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical Nucleic Acids. J. Am. Chem. Soc. 2012, 134 (3), 1376−1391. (9) Hill, H. D.; Mirkin, C. A. The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange. Nat. Protoc. 2006, 1 (1), 324−336. (10) Macfarlane, R. J.; O’Brien, M. N.; Petrosko, S. H.; Mirkin, C. A. Nucleic Acid-Modified Nanostructures as Programmable Atom Equivalents: Forging a New ‘Table of Elements’. Angew. Chem., Int. Ed. 2013, 52 (22), 5688−5698. (11) Herne, T.; Tarlov, M. Characterization of DNA probes immobilized on gold surfaces. J. Am. Chem. Soc. 1997, 7863 (119), 8916−8920. (12) Cutler, J. I.; Zheng, D.; Xu, X.; Giljohann, D. A.; Mirkin, C. A. Polyvalent Oligonucleotide Iron Oxide Nanoparticle “Click” Conjugates. Nano Lett. 2010, 10 (4), 1477−1480. (13) Cutler, J. I.; Zhang, K.; Zheng, D.; Auyeung, E.; Prigodich, A. E.; Mirkin, C. A. Polyvalent Nucleic Acid Nanostructures. J. Am. Chem. Soc. 2011, 133 (24), 9254−9257. (14) Seferos, D. S.; Prigodich, A. E.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Polyvalent DNA Nanoparticle Conjugates Stabilize Nucleic Acids. Nano Lett. 2009, 9 (1), 308−311. (15) Kwak, M.; Herrmann, A. Nucleic Acid/Organic Polymer Hybrid Materials: Synthesis, Superstructures, and Applications. Angew. Chem., Int. Ed. 2010, 49 (46), 8574−8587.
4. CONCLUSIONS The results described above provide insight into the properties of a dense shell of DNA organized around a soft bottlebrush polymeric core. The DNA remains accessible to sequencespecific restriction endonucleases as well as to stranddisplacing oligonucleotides, indicating a reasonable degree of flexibility. More surprisingly, displacing one of the two DNA strands, leaving a single-stranded DNA shell, does not lead to the dissociation of YOYO-1, a tetracationic bis-intercalating dye, even given the option of binding to dsDNA free in solution. Thus, the DBBP’s densely localized DNA shell has enhanced interactions such as with intercalating positively charged dyes. However, these DBBPs simultaneously maintain the intrinsic features of DNA such as the ability to undergo strand displacement reactions and accommodate enzyme binding and activity. This duality likely arises from the flexible polymer backbone that can provide variability to the local density of the DNA.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b02038.
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REFERENCES
Structures and synthesis of the DNA bottle brush polymers, DNA sequences used, synthesis of the DNA− brush polymer conjugate, hybridization of the complementary strand with the DNA−brush polymer conjugate, YOYO-1 fluorescence that is similar for DNA attached to the polymer brush or free in solution, and fluorescence measurements on duplex DNA−brush polymer conjugate or (nonbrush) DNA (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel: (412) 268-4196. E-mail:
[email protected]. *Tel: (412) 268-6871. E-mail:
[email protected]. ORCID
Bruce A. Armitage: 0000-0003-0109-1461 Subha R. Das: 0000-0002-5353-0422 F
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(36) Zhang, D. Y.; Winfree, E. Control of DNA Strand Displacement Kinetics Using Toehold Exchange. J. Am. Chem. Soc. 2009, 131 (47), 17303−17314. (37) Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. Nano-Flares: Probes for Transfection and mRNA Detection in Living Cells. J. Am. Chem. Soc. 2007, 129, 15477−15479. (38) Halo, T. L.; McMahon, K. M.; Angeloni, N. L.; Xu, Y.; Wang, W.; Chinen, A. B.; Malin, D.; Strekalova, E.; Cryns, V. L.; Cheng, C.; Mirkin, C. A.; Thaxton, C. S. NanoFlares for the detection, isolation, and culture of live tumor cells from human blood. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (48), 17104−17109. (39) Yang, Y.; Huang, J.; Yang, X.; Quan, K.; Wang, H.; Ying, L.; Xie, N.; Ou, M.; Wang, K. FRET Nanoflares for Intracellular mRNA Detection: Avoiding False Positive Signals and Minimizing Effects of System Fluctuations. J. Am. Chem. Soc. 2015, 137 (26), 8340−8343. (40) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.; Mathies, R. A.; Glazer, A. N. Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res. 1992, 20 (11), 2803− 2812.
(16) Averick, S.; Paredes, E.; Li, W.; Matyjaszewski, K.; Das, S. R. Direct DNA Conjugation to Star Polymers for Controlled Reversible Assemblies. Bioconjugate Chem. 2011, 22 (10), 2030−2037. (17) Averick, S.; Dey, S. K.; Grahacharya, D.; Matyjaszewski, K.; Das, S. R. Solid-Phase Incorporation of an ATRP Initiator for Polymer−DNA Biohybrids. Angew. Chem., Int. Ed. 2014, 53 (10), 2739−2744. (18) Alemdaroglu, F. E.; Alemdaroglu, N. C.; Langguth, P.; Herrmann, A. Macromol. Rapid Commun. 2008, 29 (4), 326−329. (19) Williford, J.-M.; Santos, J. L.; Shyam, R.; Mao, H.-Q. Shape Control in Engineering of Polymeric Nanoparticles for Therapeutic Delivery. Biomater. Sci. 2015, 3 (7), 894−907. (20) Fouz, M. F.; Mukumoto, K.; Averick, S.; Molinar, O.; McCartney, B. M.; Matyjaszewski, K.; Armitage, B. A.; Das, S. R. Bright Fluorescent Nanotags from Bottlebrush Polymers with DNATipped Bristles. ACS Cent. Sci. 2015, 1 (8), 431−438. (21) Mukumoto, K.; Li, Y.; Nese, A.; Sheiko, S. S.; Matyjaszewski, K. Synthesis and Characterization of Molecular Bottlebrushes Prepared by Iron-Based ATRP. Macromolecules 2012, 45 (23), 9243−9249. (22) Beers, K. L.; Gaynor, S. G.; Matyjaszewski, K.; Sheiko, S. S.; Möller, M. The Synthesis of Densely Grafted Copolymers by Atom Transfer Radical Polymerization. Macromolecules 1998, 31 (26), 9413−9415. (23) Paredes, E.; Das, S. R.; Paredes, E.; Das, S. R. Optimization of Acetonitrile Co-solvent and Copper Stoichiometry for PseudoLigandless Click Chemistry with Nucleic Acids. Bioorg. Med. Chem. Lett. 2012, 22 (16), 5313−5316. (24) Averick, S. E.; Paredes, E.; Dey, S. K.; Snyder, K. M.; Tapinos, N.; Matyjaszewski, K.; Das, S. R. Autotransfecting Short Interfering RNA through Facile Covalent Polymer Escorts. J. Am. Chem. Soc. 2013, 135 (34), 12508−12511. (25) Matyjaszewski, K.; Xia, J. Atom Transfer Radical Polymerization. Chem. Rev. 2001, 101 (9), 2921−2990. (26) Matyjaszewski, K. Atom Transfer Radical Polymerization (ATRP): Current Status and Future Perspectives. Macromolecules 2012, 45 (10), 4015−4039. (27) Matyjaszewski, K.; Tsarevsky, N. V. Macromolecular Engineering by Atom Transfer Radical Polymerization. J. Am. Chem. Soc. 2014, 136, 6513−6533. (28) Benvin, A. L.; Creeger, Y.; Fisher, G. W.; Ballou, B.; Waggoner, A. S.; Armitage, B. A. Fluorescent DNA Nanotags: Supramolecular Fluorescent Labels Based on Intercalating Dye Arrays Assembled on Nanostructured DNA Templates. J. Am. Chem. Soc. 2007, 129 (7), 2025−2034. (29) Ö zhalıcı-Ü nal, H.; Armitage, B. A. Fluorescent DNA Nanotags Based on a Self-Assembled DNA Tetrahedron. ACS Nano 2009, 3 (2), 425−433. (30) Barnaby, S. N.; Perelman, G. A.; Kohlstedt, K. L.; Chinen, A. B.; Schatz, G. C.; Mirkin, C. A. Design Considerations for RNA Spherical Nucleic Acids (SNAs). Bioconjugate Chem. 2016, 27 (9), 2124−2131. (31) Barnaby, S. N.; Lee, A.; Mirkin, C. A. Probing the inherent stability of siRNA immobilized on nanoparticle constructs. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (27), 9739−9744. (32) Newman, M.; Strzelecka, T.; Dorner, L.; Schildkraut, I.; Aggarwal, A. Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science 1995, 269 (5224), 656−663. (33) Viadiu, H.; Aggarwal, A. K. The role of metals in catalysis by the restriction endonuclease BamHI. Nat. Struct. Biol. 1998, 5 (10), 910−916. (34) Zhang, D. Y.; Seelig, G. Dynamic DNA nanotechnology using strand-displacement reactions. Nat. Chem. 2011, 3 (2), 103−113. (35) Holloman, W. K.; Wiegand, R.; Hoessli, C.; Radding, C. M. Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination. Proc. Natl. Acad. Sci. U. S. A. 1975, 72 (6), 2394−2398. G
DOI: 10.1021/acs.langmuir.8b02038 Langmuir XXXX, XXX, XXX−XXX
