Formation of Silver Nanostructures by Rolling Circle Amplification

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Formation of Silver Nanostructures by Rolling Circle Amplification Using Boranephosphonate Modified Nucleotides Camilla Russell, Subhadeep Roy, Saheli Ganguly, Xiaoyan Qian, Marvin H. Caruthers, and Mats Nilsson Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 10 Jun 2015 Downloaded from http://pubs.acs.org on June 10, 2015

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Analytical Chemistry

Formation of Silver Nanostructures by Rolling Circle Amplification Using Boranephosphonate Modified Nucleotides Camilla Russell‡, Subhadeep Roy†, Saheli Ganguly†, Xiaoyan Qian∞, Marvin H. Caruthers†* and Mats Nilsson‡.∞*. ‡

Department of Immunology, genetics & pathology, Uppsala University, Uppsala, SE-751 85, Sweden Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, 80309, United States ∞ Science for Life laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-171 21, Sweden †

ABSTRACT: We investigate the efficiency of incorporation of boranephosphonate modified nucleotides by phi29 DNA polymerase, and present a simple method for forming large defined silver nanostructures by rolling circle amplification (RCA) using boranephosphonate internucleotide linkages. RCA is a linear DNA amplification technique that can use specifically circularized DNA probes for detection of target nucleic acids and proteins. The resulting product is a collapsed single stranded DNA molecule with tandem repeats of the DNA probe. By substituting each of the natural nucleotides with the corresponding 5’-(α-PBorano)deoxynucleosidetriphosphate only a small reduction in amplification rate is observed. Also, by substituting all four natural nucleotides it is possible to enzymatically synthesize a micrometer sized single stranded DNA molecule with only boranephosphonate internucleotide linkages. Well defined silver particles are then readily formed along the rolling circle product.

Rolling circle amplification (RCA) is an isothermal enzymatic DNA polymerization reaction that produces long single stranded DNA molecules with tandem repeats of the amplified circular sequence.1,2 The circle is amplified using phi29 polymerase in a highly processive DNA replication reaction3 which has a rate of about 90kb DNA per hour.1 These long single stranded products spontaneously form random coils with a diameter of about 700 nm after 1h of RCA.4,5 RCA in combination with padlock probes has two interesting areas of application. The first is to assemble structures6 and the second is a sensor platform,7-9 where many applications and uses have been shown since its introduction in 1998.1,2 During the last 10 years various sensor techniques using RCA have been published; several fluorescence based techniques such as in situ detection,10 digital quantification11,12 and array based methods 13,14 but also colorimetric,15 magnetic16,17 and electrical6,18 sensor concepts. Lately, RCA has also received attention for its applicability in the synthesis of nanostructures,7 more recently DNA origami.19 A recently developed technique to metallize DNA is the use of 2’-deoxyoligonucleotides comprised of boranephosphonate internucleotide linkages (bpDNA). In bpDNA one of the nonbridging phosphate oxygens is replaced by a borane (BH3) group, which is able to reduce metal ions such as Au3+, Pt2+ and Ag+ to produce the corresponding nanoparticles.20 Roy et. al.21 have shown that this process can be used to construct silver nanoassemblies with high spatial resolution. The ability to incorporate 5’-(α-P-Borano)deoxynucleosidetriphosphates (dNTPαBs) (Figure 1) into rolling circle products using phi29 polymerase would open up new potentials and, for many of the developed nanotechnology and sensors techniques based on RCA, be an advantage. This is due to the formation of

metal nanoparticles by the dNTPαBs that enables multi-modal detection including optical such as fluorescence, light scattering, fluorescence and visual, and electrochemical. Two examples where this would be an advantage are in the construction of gold nanowires using RCA for electrical detection of DNA,6 and in the formation of metal nanostructures by the folding of DNA origami nanostructures using ‘monoclonal stoichiometric’ single-stranded DNA oligonucleotides produced by RCA.19 DNA can be metallized either by the reduction of metal ions such as silver22,23, gold24,25 and palladium26,27 bound to the DNA backbone using reducing agents such as sodium borohydride, or by functionalized gold nanoparticles (AuNP) containing a sequence that is complementary to a specific site of the DNA assembly. Both methods have some limitations discussed by Roy et.al.21 Reduction of metal ions bound to the DNA backbone is more applicable to simple nanostructures and requires double stranded DNA. For functionalized AuNP the smallest gap achieved is 20-30 nm due to interparticle repulsions.28,29 In this paper we investigate boranephosphonate internucleotide linkages in rolling circle products as an alternative method of metallization. We first determine the rate, using two independent methods, at which dNTPαBs (Figure 1) are incorporated during RCA using the phi29 DNA polymerase. Subsequently, we determine the degree of metallization of the rolling circle products (RCPs) where two, three and four nucleotides have been exchanged to dNTPαB. With this metallization method it will be possible in a very simple and controlled metallization step to form regularly spaced metal nanoparticles with a gap much smaller than 20 nm, which is an advantage when constructing nanowires or other metal nanostructures.

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EXPERIMENTAL SECTION Preparation of boranophosphonate nucleotides. dNTPαBs are synthesized and purified according to a previously reported method.30 This synthetic method produces a mixture of diastereomeric dNTPαB (Figure 1) that are separated by reverse phase HPLC on a C-18 column using a gradient of acetonitrile and 0.05 M triethylammonium bicarbonate. One of the two eluted diastereomers was expected to be more efficiently incorporated than the other when using phi29 DNA polymerase. To determine which of the eluted diastereomers has higher incorporation efficiency, we investigate the rate at which both isomer of each nucleotide is incorporated during the polymerization reaction.

Figure 1. The chemical structure of 5’-(α-PBorano)deoxynucleosidetriphosphates (A), where (B) is the Rp and (C) is the Sp configuration of dNTPαB. R is pyrophosphate.

Preparation of rolling circle products for digital quantification. 100 nM padlock probes are phosphorylated in PNK buffer (50 mM Tris-HCl (pH 7.6 at 25°C), 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine) with 1 mM ATP and 0.1 units of T4 polynucleotide kinase at 37°C for 30 min and then at 65°C for 20 min. For hybridization and ligation of the phosphorylated padlock probe, 100 pM of the padlock probe is incubated with 300 pM ligation template in 1xPhi29 polymerase buffer (33 mM Tris-acetate (pH 7.9 at 37°C), 10 mM Mgacetate, 66 mM K-acetate, 0.1 % (v/v) Tween 20, 1 mM DTT) with 1 mM ATP, 0.2 µg/µl BSA and 0.02 U/µl T4 DNA ligase at 37°C for 15 min. Rolling circle amplification is performed for 1 h at 37°C using 10 pM ligation mix, 1xPhi29 buffer, 0.125 mM boranophosphate modified dNTP, 0.2 µg/µl BSA and 0.08 U/µl phi29 polymerase. As a control, normal dNTPs were used. The RCA products are then labelled with a fluorescently tagged oligonucleotide in a hybridaization buffer (5 nM detection probe, 1 M NaCl, 20 mM EDTA, 20 mM Tris-HCl pH 8 and 0.1 % (v/v) Tween 20) at 70°C for 2 min and then at 55°C for 15 min. Preparation of rolling circle products for real-time RCA measurements. Phosphorylation and ligation was completed as outlined in the previous section with a modification of using 10 nM padlock probe and 30 nM ligation template. RCA using 3 nM – 0 nM ligation mixture, 1xPhi29 buffer, 0.125 mM dNTPs, 0.2 µg/µl BSA, 1xSYBR Green II and 0.1 U/µl phi29

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polymerase was performed for 4 h at 37°C where fluorescence measurements were taken every minute using a QPCR machine. Metallization. For metallization, 5 samples were prepared using dCTPαB, dGTPαB, dATPαB and dTTPαB. Two samples had two boranephosphonate modified nucleotides, one had three and one had all four nucleotides substituted with boranephosphonate modified nucleotides. A negative control containing only natural dNTPs was also tested. The samples were amplified for 2 h at 37°C in order to ensure that the sample containing all four dNTPαBs formed a good sized product. The expectation is that the reaction will proceed very slowly and have a combined effect of the rates from the dNTPαBs. These products were purified on a G-25 size exclusion column (GE Healthcare) to remove unincorporated dNTPαBs and diluted 100 fold in a 0.5xTris.acetate-EDTA buffer (pH 7.9). Long single stranded DNA molecules adopt a compact globular structure that makes it difficult to discern them by AFM or EM techniques.31,32 Thus we added three short unmodified DNA oligomers that were complementary to regions of the RCPs to the solution of the purified RCA mixture and annealed them by heating to 95°C and allowing them to cool to room temperature. This procedure converts the RCPs into partially double stranded structures that would be expected to be in a more uncoiled configuration. These RCPs were then deposited onto a 400 mesh formvar coated gold TEM grid that had been glow discharged. The grids were floated on a droplet of solution containing 0.5 mM AgNO3 in 0.5xTris.acetateEDTA buffer (pH 7.9) and incubated in this manner in a dark, humidified chamber for 20 hours at room temperature. Lastly the grids were rinsed with Millipore water and air-dried. RESULTS AND DISCUSSION Characterization of two independent assays to measure RCA rate. RCA (Figure 2) is performed in solution where circles are generated in a combined hybridization and ligation step using T4 DNA ligase. The circles are formed by the headto-tail hybridization of a padlock probe to a synthetic DNA template. For RCA, the template initiates the polymerization reaction in a buffer solution containing phi29 polymerase and free nucleotides. The resulting long single stranded product will then contain tandem repeats of the padlock probe. For every repeat, a short complementary detection oligonucleotide containing a fluorophore can be hybridized, generating a strongly concentrated fluorescently labelled globular structure due to the RCP’s thermodynamically favorable random coil form.4 The rate, at which the dNTPαBs are incorporated into the RCPs using phi29 DNA polymerase, is determined using two methods. The first method, which is an indirect measurement, uses digital quantification by fluorescence microscopy 11 and takes advantage of the strongly fluorescently labelled globular


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structures. Due to the concentration of fluorophores within the RCP, these can easily be distinguished from the background. As the RCPs pass a laser in a fluidic channel the fluorescently labelled products are registered and counted. The second method, which is a more direct measurement uses real-time monitoring of RCA with the intercalator dye SYBR Green II.33 The amount of fluorescence, which increases with increasing amount of bound SYBR Green II as the polymerization continues, is measured continuously using a real-time PCR instrument. Digital quantification is a very accurate way

of quantifying the amount of RCPs with high sensitivity and good dynamic range. However, the method does not measure polymerization rate as such, but rather the increasing detectability with increasing size of the product, which increases linearly with polymerization time.5 Hence, the direct measurement might be more ideal, but the effectiveness of the binding of SYBR Green II to different natural and modified nucleotides can influence the results. Thus, we decided to use both methods to independently generate data on polymerization rate for cross-validation.

Figure 2. Schematic illustration. For rolling circle amplification a padlock probe and a single stranded oligonucleotide are hybridized and ligated in a juxtaposed position on a template that primes the amplification using the phi29 polymerase. The collapsed rolling circle products are labelled with either SYBR Green II or a complementary detection oligonucleotide containing the fluorophore Cy3. For determining the rate of the amplification when using boranephosphonate modified nucleotides each natural dNTP is exchanged for corresponding dNTPαB in turn. The rate is also investigated when two, three or four of the natural dNTPs are exchanged for dNTPαB resulting in an increasing fraction of boranephosphonate internucleotide linkages within the RCP.

We first demonstrate, using natural dNTPs, that digital quantification can be used as an indirect method to estimate the rate at which dNTPαBs are incorporated when using phi29 polymerase. Figure S1 illustrates a linear relationship between counted rolling circle products and amplification time. Thus, the relative DNA polymerization rate can be determined by

correlating the counted globular structures obtained when using dNTPαBs to the amount of counted globular structures obtained when using natural dNTPs for the same amplification time. Real-time monitoring of RCA is a method that can be used to study the kinetics of the polymerization reaction.33 Here we



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use this method to determine the rate of RCA when using dNTPαBs and phi29 polymerase. We use the same approach to quantify the rate of RCA as Nilsson et. al.33 with the modification that we use the intercalator dye SYBR Green II as the fluorescent reporter probe. Since the RCA is a linear amplification reaction, it is possible to estimate the rate of polymerization when using dNTPαBs by measuring the steepest slope of fluorescence increase during the reaction, and relate it to a standard curve generated from a dilution series of circles using natural dNTPs (Figure S2). For both methods we determine the rate for both diastereomers from at least three experiments of triplicates. Determining the rate of RCA when using dNTPαB. First, we determined the rate of RCA for each of the dTTPαB diastereomers as well as the mixture of the diastereomers. We also investigated the dependence of the polymerization reaction on the concentration of the dTTPαB. It is important to note that, when evaluating the four different dNTPαB, each natural nucleotide is fully exchanged for a boranophosphonate modified nucleotide. Figure 3 illustrates digital quantification when using different concentrations of natural dTTP, mixture of both diastereomers for dTTPαB, and the two purified diastereomers. The rate of RCA when using normal dNTPs is constant across the concentration range of dTTP. For the 1st eluted diastereomer and the mixture of isomers, the rate of RCA increases slightly with increasing amount of dTTPαB up to a concentration of 0.125 mM. For the 2nd eluted isomer, the rate is significantly lower than for the 1st eluted isomer and the rate increases with increasing amount of dTTPαB up to 0.250 mM.

Figure 3. Digital quantification of amplification rates when using different concentrations of natural dTTP, both isomers of dTTPαB and the two purified diastereomers of dTTPαB.

For determining the rate of the three other boranephosphonate modified nucleotides we use a concentration of 0.125 mM of the modified nucleotide, which is the usual concentration of nucleotides used for RCA carried out in solution and the optimal for the 1st eluted isomer. We then determined the polymerization rate for both stereoisomers of all dNTPαB using both the direct and indirect method. The results, using both methods for determining the incorporation efficiency and amplification rate, for the four modified nucleotides using both purified forms, Table 1, show very similar outcomes. The rates are different depending on which nucleotide is being incorporated.

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Overall, the 1st eluted isomer is much more efficiently incorporated than the second for all four dNTPαBs. dGTPαB and dTTPαB have similar slight decrease in incorporation rate by phi29 polymerase, around 70-75% of the rate of natural dNTPs. The rate at which dATPαB is incorporated is only around 15% of the normal rate, while the rate for dCTPαB is about 180% of the normal rate. The padlock probe acting as the circular template during RCA does not have an equal proportion of bases, i.e. A, C, T and G are not present at 25% each. When the proportion of each of the bases is taken into account the rates alter slightly from around 70% to almost 80% for dGTPαB, from around 75% to almost 65% for dTTPαB, from 15% to 17% for dATPαB and from 180% to almost 130% for dCTPαB. These rates are consistent between different batches of synthesized dNTPαBs (not shown). Overall, the rate is 1.3-, 1.6- and 6-fold worse than the unmodified nucleotide triphosphates when using dGTPαB, dTTPαB and dATPαB respectively while 1.3-fold better when using dCTPαB. The rate of polymerization depends on the time it takes to insert the correct nucleotide, plus delays due to edit and proofread for incorrect nucleotide and the assembly and disassembly of the enzyme/ template complex. The insertion time of the nucleotide also depends on the type of nucleotide to be inserted.34 It is also likely that the incorporation rate is sequence dependent, i.e. the base ahead and after affects the rate at which the nucleotide is incorporated.34 Further research is however needed to fully understand the differences in incorporation efficiency obtained for the four dNTPαBs. In general, an average of 1.2-fold decrease, or 1.4-fold decrease when adjusted for base content, is seen which makes phi29 a more suitable polymerase to use for incorporation of dNTPαBs compared to other tested DNA polymerases. Earlier work by Shaw et. al. who first synthesized dNTPαBs35,36 demonstrated that these modified triphosphates are on average incorporated 3- and 5- fold slower when using Klenow fragments and Taq polymerase respectively when compared to natural dNTPs.37 We further investigated whether RCA is possible when more than one natural dNTP is exchanged for a dNTPαB, as depicted in the schematic illustration in Figure 2. The frequency of the boranephosphonate internucleotide linkages in the RCPs is dependent on the sequence and on the number of substituted nucleotides. Figure 4 shows real-time measurements of RCA when 2, 3 and 4 nucleotides have been exchanged. As observed, the rate decreases when the number of exchanged nucleotides increases, which is to be expected. It also appears that the SYBR II dye is less efficiently incorporated in the boranephosphonate substituted product strands, or quenched when bound, since the maximum value at saturation decreases as a function of number nucleotides substituted. Additionally, this experiment demonstrates that a completely boranephosphonate substituted DNA strand can be enzymatically synthesized using phi29 DNA polymerase. Wan and Shaw 38 have used T7 RNA polymerase to synthesize short (20-mer) RNA oligomers containing boranephosphonate linkages. However this is the first demonstration of enzymatic synthesis of a long fully substituted DNA strand.


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Table 1. The rate of RCA using the four different dNTPαB relative to natural dNTPs. The rates were measured using the direct method of monitoring SYBR green II during amplification and the indirect method of digital quantification by fluorescence microscopy. Phi29 rate of boranophosphonate modified nucleotides in relation to natural nucleotides [%] Direct 1st eluted isomer

Indirect

2nd eluted isomer

1st eluted isomer

2nd eluted isomer

70 ± 3

49 ± 6

dGTPαB

67 ± 5

50 ± 2

dCTPαB

183 ± 3

14 ± 2

177 ± 4

28 ± 4

dATPαB

14 ± 2

6±1

15 ± 1

4±2

dTTPαB

74 ± 3

1±1

79 ± 6

1±1

When adjusted for base content in product Direct 1st eluted isomer

Indirect 1st eluted isomer

dGTPαB

77 ± 5

81 ± 3

dCTPαB

131 ± 3

127 ± 4

dATPαB dTTPαB

16 ± 2

17 ± 1

62 ± 3

66 ± 6

Base content in product: G 29%, C 18%, A 29% and T 21% phate group as the metal ion is reduced. The metal being either Au(III), Pt(II) or Ag(I).

Figure 4. Real time RCA measurements where the amount of fluorescence increases as the rolling circle products grow due to the intercalation of the dye SYBR Green II. The graph illustrates successful RCA where two, three, and four natural nucleotides have been exchanged for boranephosphonate modified 2’deoxynucleotides.

Figure 6 shows micrographs of completely dNTPαBs substituted RCPs. Objects with a number of shapes are seen that appear to be partially coiled DNA structures decorated with metallic nanoparticles. RCPs normally form irregular structures where some may form several clusters rather than one globular structure.39 In this paper, the RCPs are annealed to four short complementary oligonucleotides to further open the complex, forming the loosely coiled DNA structures seen in Figure 6 and 7 and also in Figure S3 - S6. These experiments demonstrate that the boranephosphonate linked DNA molecule produced from the RCA reaction was able to reduce Ag(I) to metallic nanoparticles. Similar observations of distinct and relatively homogenously sized metallic nanoparticles were made with RCPs that were produced using only three substituted dNTPs (Figure S3). In contrast (Figure 7), larger metallized structures were observed when only two of the four nucleotides were substituted.

Metallization.

Figure 5. Simplified reaction scheme for metallization in water. The metal particle is formed in the vicinity of the boronated phos-



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Figure 6. TEM images of RCA product, where all four natural dNTPs are exchanged for dNTPαB, annealed with complementary DNA strands followed by Ag+ treatment. Images show partially coiled RCA product with metal deposition.

Figure 7. TEM images of RCA product, where (A) dCTP and dTTP are exchanged for dCTPαB and dTTPαB and (B) dGTP and dTTP are exchanged for dGTPαB and dTTPαB, annealed with complementary DNA strands followed by Ag+ treatment. Images show partially coiled RCA product with irregular metal deposition.

We have previously observed that formation of silver deposits on two dimension DNA nanostructures occurs with welldefined spatial organization only when the boranephosphonate groups were located close to each other.21 When boranephosphonate groups were separated by phosphate linkages, no detectable metallization was observed.21 Use of higher temperatures or higher concentrations of silver ions led to randomly spaced and non-specific patterns of metal deposition. In the present case, because the RCPs are inherently more flexible, when compared to our previous experiments with 2D DNA arrays, it is possible that boranephosphonates that are

located distal to each other in the sequence context are able to approach each other through coiling of the DNA strand. Such interactions would therefore lead to certain ‘hot-spots’ for metal depositions as seen in Figure 7. Image analysis (Figure 8) further demonstrates the formation of small regular metallic particles along the DNA when all four and when three out of the four natural dNTPs are substituted with boranophosphonate modified nucleotides. The analysis also shows that the metallic particles formed, when three nucleotides are exchanged, are slightly larger than when all four nucleotides are exchanged. Even larger and more


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irregularly size distributed silver particles are seen when only two of the four natural dNTPs are exchanged (Figure 8). The difference in the size of nanoparticles observed is likely explained by the fact that the fully boronated RCP has a greater number of sites of nucleation leading to a more uniform distribution of particles. With decreasing number of boranephosphonate linkages the nucleation only occurs at a few sites and the subsequent growth is concentrated at these locations. Thus for future applications it may be advantageous to use fully bornated DNA to produce a more uniform quality nanowires and nanostructurs. More images of metallized RCPs can be seen in supplementary information.

With this paper we demonstrate the possibility of forming 3D silver nanostructures by RCA using boranephosphonate modified nucleotides. This has the possibility to enable simple and efficient metallization of 1D, 2D and 3D scaffolds in a controlled manner. We believe that this will have an impact in the growing field of nanotechnology and in many sensor techniques based on RCA.

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected]

Note The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was performed within Uppsala Berzelii Technology Centre for Neurodiagnostics, financed by the Swedish Governmental Agency for Innovation Systems, the Swedish Research Council, and Uppsala University.

ASSOCIATED CONTENT Supporting Information Available Figure 8. Image analysis reveals the formation of small and narrow size distributed silver particles when all four natural dNTPs are exchanged for dNTPαB. Similar trend is observed when three of the four nucleotides are exchanged, although the particles are slightly larger. The biggest difference is seen when only two nucleotides are exchanged for the corresponding dNTPαB. Here, larger and more irregular size distributed metallic particles are formed.

The metallization procedure used in this study was not optimized for RCPs. It is 20 h at room temperature which was found to be necessary for DNA arrays,21 but this can be shortened for the RCPs. For example, use of other metals such as gold, will also enable shortening of the time as gold is reduced almost instantaneously.20 CONCLUSIONS We herein show that the rate of rolling circle amplification using 5’-(α-P-Borano) deoxynucleoside-triphosphates (dNTPαBs) is variable for the four nucleotides, but generally slightly slower compared to natural dNTPs, which is verified by two independent assays. The rate for each of the four boranephosphonate modified nucleotides differs where dCTPαB has a 1.3-fold increase while dGTPαB, dTTPαB and dATPαB have a 1.3-, 1.6 and 6-fold decrease in rate, respectively. We also demonstrate the formation of an all boranephosphonate internucleotide linked DNA molecule using phi29 polymerase. Metallization using a silver nitrate solution forms well defined silver particles along the rolling circle product that were distributed throughout the DNA templates when three and four nucleotides have been exchanged for boranephosphonate modified nucleotides. On the other hand when metallization was carried out on RCPs produced using only two of the four nucleotides; we observed the formation of larger silver particles with an irregular distribution.

Standard curves for the characterization of the two independent assays to measure RCA rate, images of metallized RCPs and oligonucleotide sequences. This information is available free of charge via the Internet at http://pubs.acs.org/.

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