Patterned DNA Metallization by Sequence-Specific Localization of a

Per Björk, Anna Herland, Ivan G. Scheblykin, and Olle Inganäs .... Biosensors and Bioelectronics 2015 74, 483-490 ..... Electronic structures of DNA...
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Patterned DNA Metallization by Sequence-Specific Localization of a Reducing Agent

2004 Vol. 4, No. 2 323-326

Kinneret Keren,† Rotem S. Berman,† and Erez Braun*,†,‡ Department of Physics, Solid State Institute, Technion- Israel Institute of Technology, Haifa 32000, Israel Received December 4, 2003

ABSTRACT Localization of a reducing agent, glutaraldehyde, on DNA molecules directs their metallization into highly conductive wires. DNA can be marked for metallization by aldehyde derivatization while retaining its biological functionality. Patterning the aldehyde derivatization of the DNA molecules in a sequence-specific manner allows to embed the precise metallization pattern into the DNA scaffold without compromising its recognition capabilities or biological functionality. We demonstrate scaffold DNA patterning by hybridization of aldehyde-derivatized and underivatized DNA molecules and by sequence-specific protection against aldehyde derivatization. This approach opens new possibilities in wiring of complex molecular-scale electronic circuits.

DNA with its remarkable molecular recognition properties and self-assembly capabilities has been proposed as a scaffold for organizing and interwiring electronic components into nanoelectronic circuits.1,2 However, the intrinsic conductivity of bare DNA is too low to allow its utilization as a molecular wire.3-5 DNA metallization has been proposed as a possible route to overcome this difficulty and convert the insulating DNA molecules into highly conductive wires.1 Several different DNA metallization schemes have been reported6 utilizing various metals, including silver,1 palladium,7 and platinum.8 In general, they consist of two steps. Metallic clusters are first formed on the DNA, and then used as nucleation sites for selective metal deposition until a continuous wire is formed. The formation of metallic nucleation centers relies on binding of metal ions or complexes to the DNA and their subsequent reduction to form metallic clusters, or on binding of small metallic particles to the DNA. These metallization schemes suffer from two major drawbacks. First, the metallization process is uniform over the entire DNA scaffold, while functional electronic circuits require controlled wiring. More importantly, metallization destroys the recognition properties of the DNA, thus preventing any subsequent biological processes. We have recently developed a method that solves the first problem by patterning the DNA metallization using a molecular lithography method, protecting specific sequences of the DNA molecules from the metallization process.9,10 Here we present * Corresponding author. E-mail: [email protected]. † Department of Physics. ‡ Solid State Institute. 10.1021/nl035124z CCC: $27.50 Published on Web 01/03/2004

© 2004 American Chemical Society

a new method that also solves the second problem; the metallization pattern is embedded into the DNA scaffold molecules by sequence-specific derivatization with glutaraldehyde, which acts a localized reducing agent on the DNA.9,10 Glutaraldehyde binding marks the DNA for metallization prior to the actual metallization process, leaving the marked DNA available for subsequent biological manipulations. Silver ions are then specifically reduced by the DNAbound aldehyde groups in the aldehyde-derivatized regions, resulting in the formation of a silver cluster chain along the DNA. An electroless gold deposition process,11 catalyzed by the silver clusters, is used to generate continuous DNAtemplated gold wires. Thus, the metallization patterning information is embedded into the scaffold DNA by aldehydederivatization without compromising the recognition properties of the DNA. This method opens new possibilities for DNA-templated electronics since it allows the construction of elaborate scaffolds using biological processes, imprinting the metallization pattern into the scaffold DNA at any stage along the way while performing the actual metallization process only as a final step. Patterning of aldehyde derivatization of DNA was achieved both by hybridization of aldehyde-derivatized and underivatized DNA molecules and by sequence-specific protection against aldehyde derivatization using homologous recombination processes by the RecA protein. DNA molecules are first aldehyde-derivatized by reacting them with glutaraldehyde (see Supporting Information). The derivatization is stable in aqueous solutions and leaves the DNA intact. Aldehyde-derivatized DNA is stretched by

Figure 1. Metallization of aldehyde-derivatized DNA. (A) and (B) SEM images of silver clusters formed along an aldehyde-derivatized DNA molecule after incubation in silver solution. Scale bars: 1 µm (A) and 300 nm (B). (C) SEM image of a DNA-templated wire after gold metallization. Scale bar: 500 nm.

combing12 on a passivated doped silicon wafer, followed by incubation in a silver solution (see Supporting Information). Positively charged silver ions are attracted to the negatively charged phosphate groups in the DNA backbone, and the DNA-bound aldehyde catalyzes their reduction, leading to the formation of metallic silver clusters along the DNA. Scanning electron microscope (SEM) images of a silver cluster chain formed on a single aldehyde-derivatized DNA molecule after incubation in the silver solution are shown in Figure 1A and 1B. The magnified image (Figure 1B) reveals some inhomogeneity in the silver cluster density along the DNA. The solid support underneath the stretched DNA molecule plays an important role in the catalysis of silver reduction by the aldehyde groups, since aldehydederivatized DNA immersed in the silver solution in bulk under the same conditions remains bare. The reduction process continues autocatalytically, as the amount of reduced silver is apparently in excess of the amount of DNA-bound aldehyde groups. Following the formation of silver nucleation sites along the DNA, a gold metallization process11 is used. In this process, gold deposition from a gold-containing complex in solution is catalyzed by the preformed silver nucleation sites, leading to the growth of metallic gold around them. This process is carried on until the gaps between silver clusters are bridged and a continuous granular wire is formed. A SEM image of the resulting gold wire is shown in Figure 1C. The need to bridge the largest gaps limits the minimal width of a continuous, few-microns-long, DNA-templated wire achievable by this approach to ∼50 nm. The wire’s length is comparable to the length of the DNA molecule in solution, but can vary due to interaction with the surface during the combing process.12 The resulting DNA-templated gold wires were shown to be highly conductive.9 Their resistivity is ∼1.5 × 10-7 Ωm, which is only 7 times larger than that of polycrystalline gold deposited by thermal or e-gun evaporation. Thinner conductive wires have not yet been demonstrated. Glutaraldehyde is known to react with primary amine groups by one of several different routes.13 We found experimentally, that unpolymerized pure glutaraldehyde is less reactive than partially polymerized glutaraldehyde. Considering this and according to ref 13, we propose that binding of glutaraldehyde to DNA occurs by a “Michaeltype” addition reaction as shown in Figure 2. Unsaturated 324

Figure 2. Proposed mechanism for aldehyde-derivatization of DNA. A primary amine group on an adenine base within a DNA molecule reacts with an R,β-unsaturated aldehyde polymer (formed by polymerization of glutaraldehyde) leading to DNA derivatization with multiple aldehyde-groups through a stable secondary amine bond. Guanine and cytosine bases also contain primary amine groups that can react in a similar manner.

polymers rather than pure glutaraldehyde react with amine groups in the DNA bases to form stable secondary amine linkage. The density of DNA-bound aldehyde is not known. A lower limit can be obtained from the density of silver clusters which can reach approximately 1 cluster per 10 nm DNA. That figure translates to at least 1 aldehyde group per 30 bp. The actual binding density may be higher. The same metallization scheme should work well with other primaryamine-containing biomolecules. For example, we have seen that RecA nucleoprotein filaments metallize in a similar manner. It seems that their metallization process is even more efficient, probably because there are more amine groups per unit length compared with DNA. A SEM image of a metallized RecA nucleoprotein filament formed by complete polymerization of RecA on DNA, followed by the standard metallization process described above, is shown in Figure S1 (Supporting Information). Other bifunctional aminereactive cross-linkers, such as bis-(sulfosuccinimidyl)suberate (BS3) and dimethyl pimelimidate (DMP), can be used instead of glutaraldehyde. For example, Figure S2 depicts a DNAtemplated wire formed using BS3 rather than glutaraldehyde. The function of DNA in various enzymatic reactions and DNA hybridization were found to be unaffected by aldehyde derivatization. We have shown that aldehyde-derivatized DNA can be used as a template for PCR amplification (Figure 3A). The RecA-promoted recombination reaction and restriction enzyme digestion operate on an aldehyde-derivatized DNA substrate (Figure 3B). We have not detected any difference in the specificity or efficiency of these reactions between bare and aldehyde-derivatized DNA. As shown below, this opens up new possibilities for patterning DNA metallization through the creation of a scaffold containing aldehyde-derivatized and underivatized regions. We have Nano Lett., Vol. 4, No. 2, 2004

Figure 3. (A) Gel electrophoresis analysis of the products of a PCR reaction designed to generate a 500 bp long fragment using λ-DNA as template. Lane 1: aldehyde-derivatized λ-DNA template. Lane 2: underivatized λ-DNA template. Lane 3 (marker): 564 bp fragment. (B) Gel electrophoresis analysis of the RecA-promoted recombination reaction and protection against restriction enzyme digestion. The recombination reaction was carried out as described in the Supporting Information using either an aldehyde-derivatized or an underivatized λ-DNA substrate and a 2114 base long probe. The reaction products were digested with HindIII restriction enzyme (NEB) in the recombination buffer for 120 min at 37 °C. HindIII has one restriction site within the probe-homologous segment on λ-DNA (this restriction site separates between the 9188 and the 2322 bp long restriction fragments). Lane 1: control reaction without RecA. Lane 2: reaction with an aldehyde-derivatized λ-DNA template. Lane 3: reaction with an underivatized λ-DNA template. Note the additional band appearing in lanes 2 and 3 (marked by arrows) owing to protection by the nucleoprotein filament. Lane 4 (marker): λ-DNA HindIII digest exhibiting the pattern expected with no protection.

Figure 4. Ligation of aldehyde-derivatized and underivatized DNA fragments. (A) Schematic illustration of the ligation reaction between a 2044 bp long aldehyde-derivatized DNA fragment and a 1096 bp long underivatized fragment with complementary sticky ends. (B) Gel electrophoresis analysis of the ligation reaction (right lane) run against a λ-HindIII digest marker (left lane). Distinct bands corresponding to 3, 4, 5, 6, 7, and 8 kbp long ligation products are clearly visible. (C) SEM images of patterned metallization of DNA molecules obtained by ligation of alternating aldehydederivatized 2 kbp fragments and underivatized 1 kbp ones. Upper image: two metallized 2 kbp (∼700 nm) aldehyde derivetized fragments separated by a 1 kbp underivatized fragment. Scale bar: 200 nm. Lower image: Three metallized 2 kbp aldehyde derivatized fragments separated by two 1 kbp fragments. Scale bar: 330 nm.

previously shown patterning of DNA metallization using RecA as a sequence-specific resist, protecting the aldehydederivatized DNA against metallization in the silver solution.9 The approach described here is advantageous since the patterning reactions are performed in bulk by marking the DNA with aldehyde prior to DNA metallization, leaving the entire DNA scaffold accessible for further biological manipulations. Two different patterning methods are presented below. Hybridization between complementary sequences has been shown to be a powerful strategy for assembling various DNA constructs.14,15 Here we show that hybridization of aldehydederivatized and underivatized DNA building blocks leads to the formation of scaffold DNA molecules with a predesigned aldehyde derivatization blueprint that is transformed into a metallization pattern. Two different DNA fragments of length 1106 and 2054 bps are generated by PCR. Restriction enzyme digestion with HgaI generates five-bases-long complementary sticky ends at both ends of these fragments as illustrated in Figure 4A. The longer fragment is derivatized with glutaraldehyde, while the shorter fragment is left underivatized. Ligation of these fragments to each other

based on their complementary sticky ends yields DNA molecules composed of alternating aldehyde-derivatized and underivatized fragments (Figure 4A). The reaction products run against a standard ruler in gel electrophoresis are depicted in Figure 4B. Bands corresponding to ligation products of length 3, 4, 5, 6, 7, and 8 kbp are resolved. These reaction products are used as scaffolds to create DNA molecules with alternating metallized and unmetallized regions. The products of the ligation reaction are purified and combed onto a passivated silicon wafer. Incubation in the silver solution leads to the formation of the corresponding metallization pattern. Figure 4C depicts two (upper) and three (lower) 2054-base-long (∼700 nm) metallized DNA fragments separated by 1106-base-long (∼350 nm) gaps. A different approach to patterning aldehyde derivatization of DNA relies on the RecA-promoted recombination reaction.16 Similar to our previous work,9,10 we utilize the RecA as a sequence specific resist. However, here the RecA is used to protect the DNA from the aldehyde derivatization rather than the metallization process itself. RecA is used to localize a 2,114 base single stranded probe molecule on a homologous section in the middle of a 48,502 base pair λ-DNA

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These results demonstrate that the new method of localizing the reducing agent in a sequence-specific manner on the DNA scaffold molecule, rather than protecting the DNA against the metallization process, leads to essentially the same results as previously shown.9,10 However, here we do not sacrifice the DNA recognition properties in the process of embedding the metallization pattern into the molecules. The metallization design of a DNA scaffold can be tailored in bulk reactions while retaining the biological functionality of the DNA and enabling subsequent manipulations. These capabilities open up new possibilities for DNA-templated electronics, such as patterning the metallization of branched DNA structures including junctions and networks, which was previously impossible. Acknowledgment. The research was conducted in the Ben and Esther Rosenbloom Nanoelectronics by Biotechnology center of excellence. Research was supported by the Israeli Science Foundation and the Technion grant for promotion of research. K.K. acknowledges support by the Clore Foundation.

Figure 5. Protection against aldehyde-derivatization of DNA using RecA as a sequence-specific resist. (A) Schematics of the protection reaction. (i) RecA monomers polymerize on a ssDNA probe molecule to form a nucleoprotein filament. (ii) The nucleoprotein filament binds to a dsDNA molecule at a homologous sequence. (iii) Incubation with glutaraldehyde leads to aldehyde-derivatization of the substrate molecule in regions unprotected by RecA. (iv) Incubation of the patterned substrate DNA molecules in silver solution results in the formation of silver clusters in the aldehydederivatized regions, while the underivatized protected regions remain bare. (B) Atomic force microscope (AFM) image of a 2114 base RecA nucleoprotein filament bound to a λ-DNA substrate molecule. (C) SEM image of the ∼1 µm gap formed by protection against aldehyde derivatization with a 2114 base long DNA/RecA nucleoproteine filament in the middle of a λ-DNA molecule. Scale bars (B) 500 nm and (C): 400 nm.

substrate (Figure 5A(i) and (ii), 5B). Sequence specific nucleoprotein binding to the substrate molecule is verified by protection against digestion by restriction enzyme (Figure 3B). Following the recombination reaction, the molecules are reacted with gluteraldehyde. The binding of RecA to the DNA blocks its reaction with glutaraldehyde, thus creating an underivatized region on the DNA (Figure 5A(iii)). After removal of excess glutaraldehyde, the RecA protein is disassembled and the DNA purified. The resulting DNA molecules, which possess a predesigned aldehyde-derivatization pattern, are combed onto a passivated silicon wafer and incubated in a silver solution (Figure 5A(iv)). The patterned aldehyde derivatization serves as a blueprint for the creation of an insulating gap in the metallization, as shown in Figure 5C.

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Supporting Information Available: Material and methods section. Figure S1: SEM image of aldehyde-derivatized RecA nucleoprotein filament. Figure S2: SEM image of DNA metallization with BS3. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775. (2) Eichen, Y.; Braun, E.; Sivan, U.; Ben-Yoseph, G. Acta Polym. 1998, 49, 663. (3) Dekker, C.; Ratner, M. Phys. World 2001, 14(8), 29. (4) Gomez-Navarro, C.; Moreno-Herrero, F.; de Pablo, P. J.; Colchero, J.; Gomez-Herrero, J.; Baro, A. M. Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 8484. (5) Bockrath, M.; Markovic, N.; Shepard, A.; Tinkham, M.; Gurevich, L.; Kouwehoven, L. P.; Wu, M. W.; Sohn, L. L. Nano Lett. 2002, 2, 187. (6) Richter, J. Physica E 2003, 16, 157. (7) Seidel, R.; Mertig, M.; Pompe, W. Surf. Interface Anal. 2002, 33, 151. (8) Richter, J.; Mertig, M.; Pompe, W.; Monch, I.; Schackert, H. K. Appl. Phys. Lett. 2001, 78(4), 536. (9) Keren, K.; Krueger, M.; Gilad, R.; Ben-Yoseph, G.; Sivan, U.; Braun, E. Science 2002, 297, 72. (10) Keren, K.; Berman, R. S.; Buchstab, E.; Sivan, U.; Braun, E. Science 2003, 302, 1380. (11) Eichen, Y.; Sivan, U.; Braun, E. PCT WO0025136 1999. (12) Allemand, J. F.; Bensimon, D.; Jullien, L.; Bensimon, A.; Croquette, V. Biophys. J. 1997, 73(4), 2064. (13) Hermanson, G. T. Bioconjugate Techniques; Academic Press: SanDiego, 1996. (14) Seeman, N. Nature 2003, 421, 427 and references therein. (15) Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H. Science 2003, 301, 1882. (16) Cox, M. M. Prog. Nucl. Acid Res. Mol. Biol. 2000, 63, 311.

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