LETTER pubs.acs.org/NanoLett
Block Copolymer Nanoparticles as Nanobeads for the Polymerase Chain Reaction Siyan Zhang,† Robert K. Prud’homme,† and A. James Link*,†,‡ Departments of †Chemical and Biological Engineering and ‡Molecular Biology, Princeton University, Princeton, New Jersey 08544, United States
bS Supporting Information ABSTRACT: New sequencing technologies based on massively parallel signature sequencing (MPSS) have been developed to reduce the cost of genome sequencing. In some current MPSS platforms, DNA-modified micrometer-scale beads are used to template the polymerase chain reaction (PCR). Reducing the size of the beads to nanoscale can lead to significant improvements in sequencing throughput. To this end, we have assembled polymeric nanobeads that efficiently template PCR, resulting in DNAdecorated “nanobeads” with a high extent of functionalization. KEYWORDS: Block copolymers, click chemistry, massively parallel signature sequencing, self-assembly, polymerase chain reaction
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apid and low cost genome sequencing is an area of tremendous interest and investment1 because the ability to quickly decode an individual’s genome may usher in advances in personalized medicine. In the quest for more rapid and inexpensive DNA sequencing, several new technologies based on massively parallel signature sequencing (MPSS) have emerged.2,3 MPSS technologies involve reading many (often millions) short DNA sequences concurrently. In one implementation of MPSS, single DNA sequences are amplified using the polymerase chain reaction (PCR) on micrometer-scale beads.4,5 Millions of these beads, each displaying multiple copies of a single DNA sequence, are then deposited on a slide, and the sequence on each bead is determined using cycles of ligation chemistry with a fluorescent readout. This technology is currently implemented in Applied Biosystems’ SOLiD instruments. One potential method to increase the throughput of such beadbased sequencing is to miniaturize the currently used micrometer-scale beads down to the nanoscale. A 10-fold reduction in the diameter of the particles could lead to an increase in sequencing throughput up to 100-fold while retaining essentially the same reagent costs associated with the sequencing reactions. Here we describe the synthesis of PCR nanobeads: ∼100 nm block copolymer nanoparticles functionalized with singlestranded DNA (ssDNA) primers. We show that these nanobeads can efficiently template PCR resulting in particles with extensive double-stranded DNA (dsDNA) surface functionalization. We have previously described a method, flash nanoprecipitation,6,7 to generate nanoparticles from amphiphilic block copolymers such as polystyrenepoly(ethylene oxide) (PS-PEO) and polycaprolactonepoly(ethylene oxide) (PCL-PEO).8,9 Rapid mixing of an organic stream containing the copolymer and a hydrophobic cargo with aqueous streams results in the formation of nanoparticles with high cargo loadings (Figure 1). The surface functionality of the nanoparticles can be readily modified by r 2011 American Chemical Society
chemical tailoring of the terminus of the hydrophilic PEO block of the copolymers.10,11 We have previously described the synthesis of a PS-PEO-alkyne block copolymer12 (Figure 2) that can be used to form nanoparticles that can participate in the Huisgen “click” reaction between terminal alkynes and azides.1315 Here we have generated nanoparticles consisting of this PS-PEOalkyne copolymer (PS block molecular wt ∼1500, PEO block mol. wt ∼5000), PS homopolymer (1500 mol wt), and a red fluorescent dye, hostasol red16 (Figure 1). The dye is encapsulated in the core of the particles and allows for analysis of the particles by fluorometry and fluorescence microscopy. The particles were analyzed by dynamic light scattering (DLS) and found to have an average diameter of 85 nm (Figure 3A). With alkyne-decorated nanoparticles in hand, we next turned our attention to generating an azide-labeled ssDNA that can be covalently attached to the nanoparticles. A custom 41 base DNA oligonucleotide (see Supporting Information for sequence details) was ordered with a 50 amine modification. This aminossDNA was converted into azido-ssDNA via reaction with an activated azidovalerate molecule (Figure 2). Attachment of the azido-ssDNA to alkyne nanoparticles was effected via a thermal variant of the click reaction which has previously been used to attach azido DNA oligonucleotides to alkyne-functionalized surfaces.17,18 A mixture of alkyne nanoparticles (∼123 μM alkyne moieties) and azido-ssDNA (35 μM, ∼0.29 equivalents per alkyne moiety) in phosphate-buffered saline (PBS) was incubated at 70 C for 3 days followed by microdialysis of the particles against water to remove any unreacted azido-ssDNA. DLS analysis of the nanoparticles was carried out after modification with the ssDNA. A small particle diameter increase (from 85 Received: January 24, 2011 Revised: March 14, 2011 Published: March 21, 2011 1723
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Nano Letters to 105 nm) was observed after addition of the azido-ssDNA to the particles (Figure 3A). Since ssDNA has a short persistence length (∼1 nm),19 only a small increase in size was expected after attachment of ssDNA. The mean-square end-to-end distance (R) for a single-stranded DNA can be estimated using the wormlike
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chain model20 ÆR æ ¼ 2lp Rmax 2lp 2
2
Rmax 1 exp lp
!!
where Rmax is the maximum contour length of the DNA (number of bases 0.7 nm per base),21 and lp is the persistence length of ssDNA (1 nm). So for a 41 base ssDNA, Rmax is 29 nm, and R is 7 nm. Under the ideal chain assumption, the mean-square radius of gyration (Rg) of a polymer is one-sixth of its mean-square endto-end distance ÆRg 2 æ ¼
Figure 1. Schematic of the flash nanoprecipitation process. An organic stream containing the ampiphilic PS-PEO-alkyne block copolymer, hostasol red dye, and hydrophobic PS homopolymer is rapidly mixed with water streams in an impinging jet mixer to generate kinetically frozen nanoparticles encapsulating the dye and the homopolymer. The alkyne moieties are presented on the surface of the nanoparticles.
Figure 2. Clickable materials for the assembly of DNA nanobeads. The PS-PEO alkyne copolymer (top) assembles into nanoparticles that present the alkyne moiety on their surface (refer to Figure 1). Singlestranded DNA (ssDNA) bearing an azide modification on its 50 end (bottom) can be attached to the nanoparticles via the azidealkyne click reaction.
ÆR 2 æ 6
Thus the expected radius of gyration of a 41 base ssDNA is 3 nm, which should give a 12 nm increase in the diameter of the nanoparticle upon attachment. We observed a 20 nm increase in the particle diameter (Figure 3A), which is larger than the estimated increase. It should be noted that because of the short length of the ssDNA, the assumption of an ideal chain, which requires Rmax . lp, is not strictly applicable. In addition, the DLS measurements were performed in water, so there is expected to be some intrachain electrostatic repulsion due to the negatively charged phosphate groups in the DNA.22 Such repulsions have the effect of increasing the effective persistence length of the ssDNA molecules. In addition, the extent of DNA functionalization was estimated by measuring the absorbance of the DNA on the particles at 260 nm (Figure 3B). From these measurements we conclude that each particle, which is estimated to contain ∼500 molecules of the block copolymer, is functionalized with ∼100 ssDNA molecules (see Supporting Information for calculation details). This high extent of functionalization may also contribute to our observation that the particle size increase upon ssDNA functionalization is larger than expected from the wormlike chain model as discussed in the previous paragraph. Since there is heavy functionalization of the surface of the particle, individual ssDNA chains may not be able to fully collapse, thus resulting in semiextended conformation of the ssDNA on the particles.
Figure 3. Analysis of ssDNA clicked nanoparticles. (A) Dynamic light scattering plot of particle diameter before and after addition of ssDNA. The addition of ssDNA to the surface of the particles results in a diameter increase of ∼20 nm. (B) UV spectroscopy on particles before and after ssDNA addition. The peak at 260 nm corresponds to ssDNA. The 10 mm absorbance unit on the y axis corresponds to the effective absorbance for a path length of 10 mm. 1724
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Nano Letters To have utility in MPSS applications, our ssDNA-modified DNA nanobeads must be able to template multiple polymerase chain reactions in order to generate particles with extensive dsDNA functionalization. A particle with only low levels of dsDNA following the PCR will exhibit a weak fluorescence signal in subsequent sequencing reactions. Thus the major design goal for our nanobeads is to enable the presentation of abundant amounts of dsDNA on the surface of the particles. A conventional PCR was set up to which the nanobeads were added at a final concentration of ∼7 1012 particles/mL (80 μg/mL). The
Figure 4. Schematic of the polymerase chain reaction on nanobeads. Two complete cycles of PCR are shown. The top (coding) DNA strand and the forward primer are colored in blue, while the bottom strand and reverse primer are colored in red.
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PCR also contained a 681 base pair (bp) template from the gene for the human Bak protein23 that anneals to the ssDNA. In this way, the particle-attached ssDNA functions as a forward primer in the PCR (Figure 4). A reverse primer and deoxynucleotides (dNTPs) were also included in the PCR, which was catalyzed by the Taq DNA polymerase. The PCR was carried using a standard thermal cycling program consisting of 35 cycles of denaturation, annealing, and extension at 95, 52, and 72 C, respectively. Following the PCR, multiple rounds of microdialysis were carried out to ensure the removal of any nucleic acids that were not attached to the particle, such as the template and any remaining reverse primer. PCR-amplified dsDNA nanobeads were examined first by DLS and UV spectroscopy. Given that the PCR will add a ∼700 bp dsDNA to the surface of the particle coupled with the fact that dsDNA has a long persistence length (∼35 nm),24 we expected to see a large size increase in the particles following PCR. Modeling the dsDNA as a wormlike chain as discussed above, we expect the radius of gyration of a 681 bp dsDNA to be 48 nm; thus the addition of these DNA molecules to the nanobeads should result in an increase in diameter of ∼192 nm. This expectation was borne out in the DLS analysis of the amplified nanobeads (Figure 5A). The predominant peak in the DLS is at 340 nm, an increase of 235 nm over the diameter of the ssDNA nanoparticles. The minor peak in the DLS at ∼30 nm likely corresponds to polymer micelles that are formed via thermal extraction of copolymer chains from the nanoparticle.25 It should also be noted that there is a sharpening of the 340 nm peak in the DLS measurement which is expected
Figure 5. Analysis of PCR amplified DNA nanobeads. (A) Dynamic light scattering plot of the nanobeads functionalized with ssDNA (before PCR) and dsDNA (after PCR). The addition of dsDNA via PCR results in a diameter increase of ∼235 nm. The data for the before PCR nanobeads are the same data presented in Figure 3A but appear more spread out because of a different scale on the y axis. (B) UV spectrum of dsDNA labeled nanoparticles compared to spectrum of unlabeled particles. The prominent peak at 260 nm demonstrates the addition of large amounts of dsDNA to the particles. (C, D) SEM micrographs of the nanobeads before (C) and after (D) PCR. Arrows point to individual particles, and the scale bar is the same (2 μm) in both figures. 1725
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due to the monodisperse nature of the added dsDNA. The attachment of dsDNA to the surface of the nanobead is further supported by the UV spectroscopy data showing a prominent peak at 260 nm corresponding to dsDNA (Figure 5B). Using this absorbance value, we can estimate that there are ∼32 dsDNA molecules per particle, meaning that about one-third of the covalently attached ssDNA molecules have been extended to dsDNA. This level of dsDNA coverage is close to maximum dsDNA coverage allowed by geometric constraints. On the basis of the estimation that one dsDNA can occupy an area of 4πRg3/ 3, and the number of DNA molecules required to fully cover the nanoparticle surface can be estimated as 3 3 ðRafter Rbefore Þ 3 Rg
where Rafter and Rbefore represent the radius of the nanobeads after and before PCR, respectively. Thus theoretically the nanoparticle surface can be fully covered with ∼43 dsDNA molecules. The ∼32 dsDNA molecules per particle we observe in the absorbance measurement thus correspond to about 75% coverage of the total nanoparticle surface with dsDNA. To further characterize the amplified nanobeads and to confirm the sizes observed in the DLS measurments, both ssDNA nanobeads and amplified dsDNA nanobeads were examined by scanning electron microscopy (SEM, Figure 5C). The SEM images dramatically demonstrate the increase in size of the nanobeads following PCR. As an additional characterization of the amplified nanobeads, confocal microscopy images of the particles were acquired. Prior to imaging, the amplified nanobeads were treated with a solution of Hoechst 33342, a dye that fluoresces blue upon intercalation into dsDNA. In the images presented in Supplementary Figure 1 (Supporting information), the blue Hoechst 33342 fluorescence colocalizes with the red fluorescence emitted by the hostasol red dye in the core of the particles. In summary, we have used a combination of self-assembly, organic chemistry, and molecular biology to generate new, unique bioorganic hybrid nanoscale objects composed of a block copolymer core surrounded by a thick corona of dsDNA. The use of these DNA nanobeads in conjunction with sensitive imaging techniques can enable great increases in the throughput of currently used bead-based DNA sequencing approaches and bring the promise of personalized genomic medicine closer to a reality.
’ ASSOCIATED CONTENT
bS
Supporting Information. Detailed experimental procedures for PS-PEO-alkyne block copolymer and azido-ssDNA synthesis, nanoparticle assembly, covalent conjugation of azidossDNA, PCR on nanobeads and detailed calculations of ssDNA and dsDNA surface coverage, and one figure. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
Professor Douglas Adamson (University of Connecticut) for the synthesis of the PS-PEO copolymer and Professor Ronald Hart (Rutgers University) for stimulating discussions about DNA sequencing.
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’ ACKNOWLEDGMENT This work was supported by a seed grant from the NSFsupported Princeton Center for Complex Materials. We thank 1726
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