Modular-DNA-Programmed Construction of Permanent Nanoscale

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Modular-DNA-Programmed Construction of Permanent Nanoscale Cyclic Assemblies by Reaction of Covalently linked 2,5-bis(2Thienyl)pyrrole Monomers Zhijie Ma, Wen Chen, and Gary B. Schuster* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 S Supporting Information *

ABSTRACT: DNA modules that contain complementary recognition units and covalently linked 2,5-bis(2-thienyl)pyrrole (SNS) monomers spontaneously assemble in aqueous buffer solution into cyclic structures. Ligation of the DNA modules is readily accomplished by an oxidative reaction with horseradish peroxidase (HRP)/H2O2, which results in covalent bond formation between the SNS monomers to form permanent nanoscale assemblies. The flexibility of the cyclic assemblies, and their ability to form SNS-to-SNS bonds, was assessed by varying the number and location of SNS monomers on each DNA module. Efficient ligation is observed when each module contains at least two SNS monomers. Ligation efficiency is diminished when the single stranded regions of the assembly are converted to duplex DNA. The efficient ligation of the DNA structures is attributed to the self-association of the monomers due to their hydrophobicity and is enabled by the flexibility of the single-stranded regions. It was found by systematically controlling the position and number of SNS monomers that these DNA modules are efficiently ligated into cyclic assemblies when each nucleobase module contains at least two SNS monomers. This provides a method for formation of unique covalently linked DNA structures and a process that can readily ligate or cross-link DNA chemically. KEYWORDS: modular DNA assemblies, ligated DNA nanostructures, oxidative coupled modified DNA

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INTRODUCTION Because of its powerful self-recognizing and self-organizing properties, DNA is often used as a scaffold to form ordered assemblies of components such as metal ions, nanoparticles, proteins, dyes, and chromophores.1−5 DNA scaffolds provide a means for the directed self-assembly of complex objects of defined geometry and proportions.6−10 We have used this approach to create conjoined DNA-conducting polymer nanowires. Polymers of controlled length and defined composition are formed by horseradish peroxidase/hydrogen peroxide (HRP/H2O2) oxidation when monomers (aniline, for example) are constrained in the major groove of DNA by covalent linkage to a nucleobase.11−15 In a related approach, we developed a modular DNA-programmed assembly method to form cyclic nanoarrays of conducting polymers comprised of up to thirty linked 2,5-bis(2-thienyl)pyrrole (SNS) monomer units.15 In these structures, a cyclic conducting polymer was constructed from three, four, or five DNA single strand modules, each comprised of six SNS monomers linked covalently to cytosines (designated X in Figure 1) and separated by thymines. This central (XT)5X region of the module was flanked on both 5′- and 3′-sides by 12 base Watson/Crick “recognition” sequences that were uniquely © 2012 American Chemical Society

coded to enforce spontaneous assembly into cyclic arrays. Treatment of these monomer arrays with HRP/H2O2 very efficiently ligated the modules through formation of SNS-toSNS bonds and formed cyclic SNS polymers comprised of 18, 24, or 30 SNS monomer units. In the previous examination of ligation through SNS oligomerization, the monomers on each module were arranged to be contiguous, nearly overlapping neighbors. The observed high efficiency for SNS polymerization and the flexibility of DNA single strands suggested that module ligation might be accomplished by reaction of spatially separated monomers. In this work, we examine the ligation of self-assembling single stranded DNA modules linked to a variable number of SNS monomers; see Figure 1. It was found by systematically controlling the position and number of SNS monomers that these DNA modules are efficiently ligated by reaction with HRP/H2O2 into covalently linked cyclic nanoassemblies when each 35-nucleobase module contains at least two SNS monomers. This provides a method for formation of unique Received: July 6, 2012 Revised: September 13, 2012 Published: September 26, 2012 3916

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Figure 1. Structures of the SNS-modified oligomers, Caps and the DNA assemblies used in this work. temperature overnight. The ligation reaction was performed at room temperature in 1 mL of solution by addition of 5 μL of HRP (2 mg dissolved in 1 mL of nanopure water) followed by 5 μL of H2O2 (0.03%) and 5 μL of ABTS (10 μM in water). UV−vis spectra were recorded before and after the initiation of the reaction. After the reaction, hydrazine (108 mM) was added to reduce the oxidized polymer to the leuco form. Tm Measurements. Samples were prepared by hybridization of 2 μM of DNA oligomers in 10 mM of sodium citrate buffer (pH 4.5, where HRP activity is maintained) with 200 mM of NaCl. The melting curves were measured at heating and cooling rates of 1 °C/min. Tm values were determined by differentiation of the melting curves. UV− vis spectra were recorded on a Cary 1E spectrophotometer. Nondenaturing PAGE Experiments. Nondenaturing gel experiments were performed to confirm the formation of cyclic DNA structures. Typically, samples for the experiments were hybridized by annealing unlabeled (2 μM) and radiolabeled (5000 cpm) of SNSmodified DNA oligomers in pH 7.0 buffer solution containing 10 mM sodium phosphate and 200 mM NaCl (total volume 50 μL). The samples were analyzed with a 10% polyacrylamide gel in a Hoefer Vertical Slab gel unit model SE400 (Hoefer Scientific Instruments, San Francisco, CA). The wet gel was visualized by autoradiography. Denaturing PAGE Experiments. Denaturing gel experiments were performed to study the covalent linking of DNA oligomers after reaction with HRP/H2O2. The samples for analysis were prepared by hybridization of unlabeled (1 μM) and radiolabeled (5000 cpm) SNSmodified DNA oligomer in 10 mM sodium citrate buffer (pH 4.5) with 200 mM NaCl. After reaction with HRP/H2O2/ABTS at 5 °C for 30 min, the pH of the samples was raised to 8 by addition of 1 M NaOH. Then, the samples were mixed with 50 μL of denaturing loading buffer (formamide and water in 4:1 ratio), heated at 90 °C for 5 min, analyzed with a 10% denaturing 19:1 polyacrylamide gel containing urea (3.4 M), and visualized by autoradiography.

DNA structures and a process that can readily ligate or crosslink the DNA chemically to form permanent assemblies that may be useful as robust building blocks for arrays of nanocomponents.

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EXPERIMENTAL SECTION

Materials. All commercially available reagents were used without further purification. SNS containing modified DNA oligomers were synthesized as previously reported.14 T4 polynucleiotide kinase (PNK) enzyme was purchased from New England Biolabs Inc. γ-32P-ATP was purchased from MP Biomedicals. Horseradish peroxide (HRP), type II (200 units/mg) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich, St. Louis, MO. A stock solution of HRP was prepared by dissolving 2 mg of HRP in 1 mL of nanopure water. Hydrogen peroxide was purchased from Fischer Scientific, Pittsburgh, PA, and diluted with Dnase free water to 0.03% before use. Purification of Unmodified and SNS Containing DNA Oligomers. The DNA oligomers with or without SNS units were purified by reverse phase high pressure liquid chromatography (HPLC) using a Dynamax C18 column. The purified DNA was desalted with Waters Sep Pak cartridge and characterized by ESI-mass spectrometry. The concentration of the SNS-modified DNA was measured by its absorption intensity at 260 nm with every two SNS groups counted as one cytosine (because the extinction coefficient of SNS is ca. half that of cytosine) in a web-based tool: http://www.basic. northwestern.edu/biotools/oligocalc.html. Preparation of Radiolabeled DNA. The oligonucleotides were labeled at the 5′-terminus with γ-32P-ATP and T4 kinase. The radiolabeled DNA was purified by 20% PAGE. The desired bands were excised from the gel and incubated with water at 37 °C for 12 h. The DNA was precipitated from the solution by adding 10% equiv. 5 M ammonium acetate and cold ethanol (800 μL for 200 μL of supernatant). The mixture was vortexed, stored at −78 °C for 1 h, and then centrifuged for 1 h at 13 000 rpm. The supernatant was removed, and the residue DNA was washed twice with 100 μL of 80% ethanol and dried in a Speed Vac at medium heat. Suitable amounts of water were added for further experiments. Ligation. Samples were prepared by hybridizing 2 μM of the desired single-strand DNA oligomers in 10 mM citrate buffer (pH 4.5) containing 200 mM of NaCl. Hybridization was achieved by heating the samples at 85 °C for 10 min then holding them at room

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RESULTS The SNS-containing DNA oligomers shown in Figure 1 were prepared by postsynthetic modification, as previously reported;14 each module was purified by HPLC and characterized mass spectrometrically (see Table S1, Supporting Information). The SNS monomers in these oligomers are linked covalently through a trimethylene chain to N4 of a cytosine in the central 3917

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structures are stabilized primarily by the Watson−Crick base pairing of their recognition sequences. Significantly, the Tm of the three-part assembly DNA(1,2,3) is 64 °C. The 13 °C increase in Tm for the three-part assembly compared with DNA(1,2) indicates cooperative stabilization, which is expected for an ordered cyclic array. Further, the Tm of DNA(1,2,3) is 17 °C greater than that of the identical three-module assembly lacking SNS monomers (i.e., X = C,). The melting curves (see the Supporting Information) indicate that the ordered SNS array of the cyclic assembly plays a role in stabilizing the structure.15 Comparison of the melting behaviors of the other assemblies provides additional insight into the factors that stabilize DNA(1,2,3). For example assembly DNA(1,2,8) is also comprised of three modules, but in contrast to DNA(1,2,3), there are only two SNS monomers linked to module DNA(8), resulting in a noncontiguous array of monomers. The Tm of DNA(1,2,8) is 4 °C less than that of DNA(1,2,3). All of the assemblies examined show a consistent trend in melting behavior. As the number of contiguous SNS monomers decreases, so does the Tm. For DNA(4,5,9), which contains only one SNS monomer on each module, the Tm is only 4 °C greater than that of the assembly having no SNS monomers (DNA(1,2,3); X = C). For DNA(6,7,10), which has two isolated SNS monomers on each module, the Tm is 9 °C greater than that of (DNA(1,2,3); X = C). These findings suggest that the cooperative hydrophobic interactions of the SNS monomers contribute measurably to the stabilization of the cyclic assemblies. Nondenaturing PAGE. The structures of the SNS-containing DNA cyclic assemblies were also studied by nondenaturing (native) polyacrylamide gel electrophoresis (PAGE). In this experiment, duplex DNA remains intact during analysis and the rate of migration is dependent on the size of the assembly and its total charge. Modules DNA(1), DNA(4), and DNA(6) were radiolabeled at their 5′ termini with γ-[32P] to enable visualization of the gels by autoradiography. Figure 2 shows an analysis of the assemblies formed from (DNA(1*,2,3); X = C) where the asterisk indicates the position of the radiolabel. Lane 1 shows the location on the gel of the single radiolabeled module (DNA(1*); X = C). With two modules, (DNA(1*,2);

segment of the DNA strand. For example, DNA(1) contains six SNS monomers arranged as 5′-(XT)5X-3′ with flanking recognition sequences 5′-AGCGCTTGGAGT and TGAGGTTCGCGA-3′. The recognition sequences of DNA(1) are complementary to corresponding sequences on DNA(2) and on DNA(3), for example. The assemblies shown in Figure 1 were designed to assess the ability of remotely positioned SNS monomers to ligate the DNA modules upon oxidation with HRP/H2O2. Specifically in the assembly DNA(1,2,8), modules DNA(1) and DNA(2) each have six SNS monomers while DNA(8) contains only two SNS monomers positioned to be adjacent to those on DNA(1) and DNA(2). DNA(1,2,9) and DNA(1,2,10), are comprised of two modules having six SNS monomers and one module with one or two remotely located SNS monomers, respectively. Similarly, assemblies DNA(4,5,9) and DNA(6,7,10) are comprised of three modules each containing one or two SNS monomers. Characterization of Modular Assemblies. The physical and chemical characteristics of the modular assemblies were investigated to determine their configuration and properties. Melting temperatures (Tm) were assessed to investigate cooperative behavior and thermodynamic stability. Nondenaturing gel electrophoresis reveals the association of the modules into assemblies. Absorption spectroscopy reveals cooperative interactions between remote SNS monomers. Melting Temperature. The Tm reflects the thermal stability of the separate modules compared with that of the intact assembly. Comparison of assemblies of different composition provides insight into the structure of the assembly and into the factors affecting its stability. In particular, thermal denaturation is a gauge of cooperative stabilization for the cyclic assemblies. The Tm of the assembly formed from three modules that are identical to those in DNA(1,2,3) but without appended SNS monomers (i.e., X = C; see Figure 1) is 47 °C under standard conditions (2 μM DNA oligomers in 10 mM sodium citrate buffer, pH 4.5, and 200 mM NaCl); see Table 1. This value Table 1. Melting Temperature (Tm) for the DNA Assemblies DNA assembly

Tm °C

15

(1) (1,2)15 (1,2,3)15 (1,2,3); X = C, pH 4.5 (1,2,3); X = C, pH 7 (1,2,8) (1,2,9) (1,2,10) (4,5,9) (6,7,10) (4,5,3); (3, X = C) (6,7,3); (3, X = C)

+ Cap Tm °C

+ HRP/H2O2 Tm °C

+Cap(2), 51 +Cap(2), 51 64 47 58 60 58 61 51 56 50 53

81 +Cap(1), 68 +Cap(1), 57

+Cap(1), 61 +Cap(1), 56

84 77 77 53, 75 75 49, 76 55, 78

serves as a baseline for examination of the affect SNS monomers have on the behavior of these assemblies. Similarly, the Tm of DNA(1) and Cap(2) (the latter is comprised of 12 bases complementary to the 5′ recognition sequence of DNA(1)) was determined as a baseline for the properties of the duplexes that form the recognition sequences of the SNScontaining modules in noncyclic assemblies. The Tm of the assembly DNA(1)/Cap(2) is 51 °C. The Tm of the two-part assembly DNA(1,2) is also 51 °C, which shows that these

Figure 2. Autoradiography of the nondenaturing PAGE analysis of the cyclic DNA. Lane 1 (DNA(1*); X = C); lane 2 (DNA(1*,2); X = C); lane 3 (DNA(1*,2,3); X = C); lane 4 (DNA(1*,2,3)/Cap(2); X = C), two equiv. of Cap(2) were added before annealing the DNA assembly. 3918

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Cap(1), which is complementary to the central segments of the DNA modules.18 Oligomerization of Assembled Modules. Efficient formation of SNS-to-SNS bonds result from the reaction of the SNS-containing modular assemblies with HRP/H2O2 and a catalytic amount of ABTS.19,20 The one-electron oxidation of SNS to form a radical cation results in rapid covalent bond formation between the 5- and 5′-positions of adjacent monomers.14,15 Bond formation between SNS monomers that are attached to different modules in an assembly results in ligation (i.e., linkage by covalent bond formation) of the DNA oligomers and the formation of permanent nanoassemblies. We examined the dependency of ligation on the number of SNS monomers and their position by examining the effect of reaction with HRP/H2O2 on the Tm of the assemblies, by absorption spectroscopy and assessing the migration of the ligated assemblies on denaturing PAGE gels. Melting Temperature. We previously reported that reaction of DNA(1,2,3) with HRP/H2O2 results in efficient ligation of its three modules by formation of a polymeric SNS ring. Characteristically, the polymerized assembly shows a single melting transition with Tm of 81 °C, which is 17 °C greater than that of the assembly before reaction with HRP/H2O2 (see Table 1). The reaction of DNA(1,2,8) with HRP/H2O2 was carried out as a test to determine if module ligation requires a contiguous array of SNS monomers on all modules. The reaction of DNA(1,2,8) with HRP/H2O2 results in a ligated assembly with a single melting transition at 84 °C. Evidently, efficient ligation is possible with modules that do not contain contiguous arrays of SNS monomers. Similar results are obtained for DNA(1,2,9) and DNA(1,2,10) where one module contains only one or two centrally placed SNS monomers, respectively. A different result is obtained from the reaction of DNA(4,5,9) with HRP/H2O2. In this case, each module contains only one SNS monomer, and the reaction product is a mixture of partially and fully ligated assemblies, as indicated by observation of two melting transitions. This interpretation is supported by results from the reaction of DNA(4,5,3) and DNA(6,7,3) where for module (DNA(3); X = C), thus making its complete ligation impossible. Here, too, two melting transitions are observed (see Table 1). Denaturing PAGE. DNA assemblies that are bound only by Watson−Crick complementary base pair association migrate as individual oligomers in a denaturing PAGE gel. Those assemblies in which ligation between SNS monomers on separate modules has occurred are permanent and will migrate as one unit in denaturing PAGE. We used this assay to determine SNS-to-SNS bond forming efficiency by monitoring the extent of module ligation. We have shown previously that treatment of assembly DNA(1,2,3) with HRP/H2O2 results in essentially complete ligation of its three modules. Figure 4 shows the results from reaction of HRP/H2O2 with the three-part assemblies DNA(1*,2,8), DNA(1*,2,9), and DNA(1*,2,10). Lane 1 shows the position of unreacted DNA(1*) labeled with 32P for visualization. Lanes 2−4 show the results of reaction of two part assemblies DNA(1*,8), DNA(1*,9), and DNA(1*,10) with HRP/H2O2. In each case, the two modules are efficiently ligated by SNS-to-SNS bond formation and migrate more slowly than the single module. This finding shows that there is sufficient flexibility in these two-part assemblies to permit SNS monomers on different modules to approach within bonding distance and then couple. Lanes 5−7 of Figure 4 show that

X = C), the rate of migration on the gel slows appreciably (Figure 2, lane 2), and with the three modules in a cyclic array, (DNA(1*,2,3); X = C), the migration rate slows further (Figure 2, lane 3). The formation of the cyclic assembly from (DNA(1*,2,3); X = C) was assessed by including an excess of Cap(2) in the reaction solution. Cap(2) is complementary to the 5′-recognition sequence of DNA(1). Figure 2 lane 4 shows that Cap(2) has no effect on the migration rate of the assembly, which is consistent with a cyclic structure. Similar experiments yielding similar results (see Figures S1 and S2, Supporting Information) were carried out with DNA(1*,2,3),15 DNA(1*,2,8), DNA(1*,2,9), DNA(1*,2,10), DNA(4*,5,9), and DNA(6*,7,10). The three part assemblies are not disrupted by the addition of excess Cap(2). These findings show the robust formation of cyclic assemblies from the encoded modules. Absorption Spectroscopy. The absorption spectra of the cyclic assemblies show evidence for electronic interaction between SNS monomers. Figure 3 shows the visible absorption

Figure 3. UV−vis spectrum of DNA(1,2,3) (black), DNA(6,7,10) (red), and DNA(4,5,9) (blue). Inset: normalized (for the number of monomers), deconvoluted (from the DNA absorption) peak for the SNS monomers in the DNA modules.

spectra of DNA(1,2,3), DNA(6,7,10), and DNA(4,5,9). An absorption band characteristic of SNS is clearly seen to the red (longer wavelength) of the DNA maximum at 260 nm. As expected, the intensity of the SNS absorption band increases with the number of SNS monomers in the assembly. Deconvolution (Figure 3, inset) of the SNS band from that of the DNA bases reveals a systematic blue shift as the number of SNS monomers per module increases. In assembly DNA(4,5,9) where X = C for DNA(5) and for DNA(9) so that the assembly contains only one SNS monomer, the SNS absorption band has a maximum at 347 nm. The maximum of the SNS band in DNA(4,5,9) where there are three SNS monomers, one SNS monomer per module, is 342 nm. In DNA(6,7,10), having two SNS monomers per module, this band maximum shifts to 337 nm, and for DNA(1,2,3) with six SNS monomers per module, the maximum for this band is found at 333 nm. Cap(1) (see Figure 1) is an 11 base single stranded oligomer with a sequence that is complementary to the central segments of the DNA modules bearing SNS groups. When Cap(1) is incorporated in assembly DNA(6,7,10), the SNS absorption maximum shifts from 337 to 345 nm. The observed hypsochromic shift in these maxima as the number of monomers increases indicates an interaction between the SNS monomers in the assemblies that is typical of face-to-face Htype aggregation.16,17 This interaction is affected by inclusion of 3919

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Figure 4. Autoradiography of denaturing PAGE analysis of the chemical ligation of the cyclic DNA assemblies by reaction with HRP/ H2O2. Lane 1, DNA(1*) only; lane 2, DNA(1*,8) + HRP/H2O2; lane 3, DNA(1*,9) + HRP/H2O2; lane 4, DNA(1*,10) + HRP/H2O2; lane 5, DNA(1*,2,8) + HRP/H2O2; lane 6, DNA(1*,2,9) + HRP/H2O2; lane 7, DNA(1*,2,10) + HRP/H2O2.

Figure 5. Autoradiography of denaturing PAGE analysis of the ligation of cyclic DNA by reaction with HRP/H2O2. Lane 1, DNA(6*) only; lane 2, DNA(6*,7) + HRP/H2O2; lane 3, DNA(6*,7,10) + HRP/ H2O2; lane 4, DNA(6*,7,10) + HRP/H2O2 which included Cap(1) in a molar ratio of 1 DNA assembly to 3 Cap(1) DNA oligomers, and the ratio was not optimized; lane 5, (DNA(6*,7,10); X = C for DNA(10)) + HRP/H2O2.

ligation also occurs efficiently in three-part cyclic assemblies when one module does not have a contiguous array of SNS monomers. This observation is most striking for assembly DNA(1*,2,9) where reaction with HRP/H2O2 results in essentially complete ligation, even though module DNA(9) contains only one SNS monomer ostensibly positioned remotely from the SNS monomers on modules DNA(1) and DNA(2). Quite clearly, the single stranded regions of these assemblies are sufficiently flexible that the ligation reaction is not inhibited when one SNS monomer is separated from the others. The reaction of assembly DNA(6*,7,10) with HRP/H2O2 is shown in Figure 5. In this assembly there are a total of six SNS monomerstwo on the central segment of each module. Lanes 2 and 3 of Figure 5 show that there is efficient ligation for the two- and the three-module assemblies. Cap(1), the oligomer complementary to the central segment of the DNA modules, forms duplex segments by Watson−Crick binding to the cyclic assemblies. Lane 4 of Figure 5 shows that the inclusion of Cap(1) in the reaction of assembly DNA(6*,7,10) with HRP/ H2O2 partially inhibits the ligation reaction. Without Cap(1), essentially complete ligation of all three modules occurs; in the presence of Cap(1), approximately equal amounts of fully and partially ligated structures are formed. Lane 5 shows the results of a control experiment. Reaction of HRP/H2O2 with assembly DNA(6*,7,10) where DNA(10) has X = C (i.e., this module has no covalently linked SNS groups), as expected, does not yield any of the permanent, fully ligated three-module cyclic assembly. Examination of the HRP/H2O2 reaction with assembly DNA(4*,5,9), where there are a total of three SNS monomers, one on the central segment of each module results in incomplete ligation in all cases (see Figure S3, Supporting Information). These findings show that three-module assemblies having two SNS monomers per module are efficiently ligated by reaction with HRP/H2O2. However, the ligation efficiency is significantly reduced by inclusion of an oligomer complementary to the single-strand regions of the cyclic assembly. Evidently, the flexibility of the single stranded segments of the cyclic assemblies allows the SNS monomers on different modules to come together for bond formation.

Absorption Spectroscopy. The UV−vis spectra of these cyclic DNA assemblies demonstrate characteristic spectral changes upon oxidative oligomerization by reaction with HRP/H2O2. We have shown that short SNS-oligomers formed by this reaction have characteristic absorption spectra in the visible and near IR regions consistent with those expected for polaronic or bipolaronic (SNS)n species.14 The absorption spectrum of the cyclic oligomer formed by the reaction of DNA(1,2,3) with HRP/H2O2 has bands at 580 and 1070 nm, characteristic of the conducting polymer. Reaction of this polymer with hydrazine reduces it to the leuco form. Similar characteristic spectral changes occur from the reaction assembly DNA(6,7,10) with HRP/H2O2. Figure 6 shows absorption spectra of DNA(6,7,10) before and after reaction with HRP/H2O2 and after the subsequent reaction with hydrazine. As reported above, before the oxidative ligation reaction, the spectrum of DNA(6,7,10) is comprised of bands for the DNA and the SNS monomers. The addition of HRP/H2O2 results in the appearance of new absorption bands with maxima at ca. 415, 580, and 830 nm that indicate the formation of (SNS)6+• species. The addition of hydrazine to this solution causes the reduction of the radical cation species and a concomitant shift of the absorption maximum to ca. 400 nm. The reaction of assembly DNA(4,5,9) with HRP/H2O2 gives an absorption spectrum (see Figure S4, Supporting Information) with a band at 461 nm, which is characteristic of SNS radical cation.14,21 This is consistent with the results from the melting temperature and PAGE experiments that indicate incomplete ligation when there is only one SNS monomer on each module.

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DISCUSSION It is clear from the experimental results that ligation of DNA modules by SNS-to-SNS bond formation does not require a contiguous array of adjacent SNS monomers on each module. In particular, assembly DNA(6,7,10) is fully ligated with high 3920

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The reaction of the assemblies with HRP/H2O2 results in bond formation between the SNS monomers forming a conjoined SNS-DNA polymer (see the Supporting Information for a model structure of the ligated, cyclic DNA conjoined assembly). Evidence for this ligation comes from the Tm data, which shows significant increase on reaction, the denaturing PAGE experiments that show the assemblies migrate as one species after reaction with HRP/H2O2, and the absorption spectroscopy indicates formation of oligomers with the optical properties expected for conducting polymers. It is remarkable that even with only one SNS monomer per module approximately half of the assemblies are fully ligated by reaction with HRP/H2O2. With two SNS monomers per module, this reaction results in essentially complete ligation. This finding shows that the monomers self-organize before or during the oxidative coupling reaction. It is clear that ligation of separated SNS monomers into cyclic arrays22−24 requires flexibility in the DNA single strand region of the assembly. This flexibility is reduced when the complementary duplex is formed by the inclusion of Cap(1). The results of the denaturing PAGE support this finding: the inclusion of Cap(1) reduces the ligation efficiency. These PAGE results are consistent with the thermal denaturation profiles, and all indicate that the flexibility in the loop region is a crucial factor for efficient ligation. We previously showed that cyclic arrays of poly-SNS on a DNA scaffold can be readily prepared from three, four, or five module assemblies containing 18, 24, and 30 SNS monomers, respectively. In this work, we have shown that it is possible to prepare a permanent, covalently linked cyclic DNA nanoassembly containing any arbitrary number of SNS monomers greater than three. These permanent assemblies may be useful building blocks for the construction of robust two- and threedimensional structures built spontaneously from self-organizing DNA modules.

Figure 6. UV−vis absorption spectra of: DNA(6,7,10) before (blue line) and after (black line) reaction with HRP/H2O2; the reduced, leuco, (red line) formed by the addition of hydrazine after the reaction with HRP/H2O2.

efficiency even though it contains only two SNS monomers per module. However, assembly DNA(4,5,9) with only one SNS monomer per module is incompletely ligated by reaction with HRP/H2O2. Furthermore, it is found that ligation of assembly DNA(6,7,10) is partially inhibited when Cap(1) is included in the reaction mixture and the central segment is converted from a single strand to a duplex. These findings point to the important role that flexibility of the single strand segments plays in permitting the SNS monomers to come within bonding distance. This flexibility is shown schematically in Figure 7, which represents the ligation reaction of the three-module DNA

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ASSOCIATED CONTENT

S Supporting Information *

Mass spectral analysis of the DNA oligomers, autoradiography of nondenaturing and denaturing PAGE analysis of DNA assemblies, melting curves of DNA assemblies, a model of cyclic polySNS on DNA templates and UV−vis absorption spectra of DNA assemblies. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 7. Ligation reaction of the three-module DNA assemblies that do not have a continuous array of adjacent SNS monomers.

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AUTHOR INFORMATION

Notes

assemblies that do not have a contiguous array of adjacent SNS monomers. The duplex “arms” formed by Watson−Crick association of the recognition sequences are depicted as red, green, and blue linear regions. The SNS monomers, depicted as purple spheres, are attached to the flexible single strand and self-associate before reaction with HRP/H2O2. Evidence for this self-association comes both from the Tm data and from the absorption spectroscopy experiments. The Tm data show that the stability of the assemblies increase with increasing number of SNS monomers and the absorption spectroscopy reveals a hypsochromic shift typical of H-aggregation. It is not surprising that the SNS monomers self-associate in aqueous media when that is possible because of their extensive hydrophobic surface. Evidently, the single strand region of the DNA assembly is sufficiently flexible to permit the SNS monomers to stack at least partially.

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Vasser Woolley Foundation. REFERENCES

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Chemistry of Materials

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dx.doi.org/10.1021/cm302104z | Chem. Mater. 2012, 24, 3916−3922