An Engineered DNA‐Binding Protein Self‐assembles Metallic Nanostructures

Biological fabrication routes can provide a way to overcome the limitations presented by current chemistry-based nanoparticle arrangement and assembly methods. Many recent assembly strategies utilize DNA as the templating molecule by patterning gold nanoparticles on DNA through chemical conjugation via, for example, a sulfhydryl bond. Reliance upon this chemistry, however, limits applications because it acts indiscriminately on several different metals and is only useful for some noble-metal nanoparticles. Strategies that covalently link nanoparticles to proteins or DNA risk denaturation or distortion of native protein, distortion of DNA, and/or disruption of the plasmonic or photonic properties unique to nanoparticles. We present a strategy for nanoparticle patterning on DNA that utilizes the biologically based self-assembly properties of DNA-binding proteins to facilitate the targeted immobilization of nanoparticles on DNA. Here we show that a derivative of the DNA-binding protein TraI spontaneously organizes colloidal gold nanoparticles on DNA through an engineered gold-binding peptide motif. This system, based solely on specific, noncovalent, biologically determined interactions, represents significant progress on the route to spontaneously ordered assembly of nanoparticles important for downstream applications in nanoelectronics and photonics. TraI (192 kDa, 1756 residues) is the E. coli F-plasmid-encoded relaxase/helicase that harbors sequence-specific single-stranded-DNA-binding activity (relaxase domain) and nonspecific single and double-stranded-DNA-binding activity (helicase domain). We engineered TraI with a gold-binding motif at an internal permissive site after residue Q369 to direct the assembly of gold nanoparticles (AuNPs) on DNA through noncovalent interactions. Permissive sites, regions of proteins that tolerate a wide variety of amino acid additions without disrupting native protein function, were previously identified through transposon/epitope tag mutagenesis in TraI. This study utilizes TraI’s nonspecific DNA-binding activity as the first step toward optimization of this biologically based nanoparticletemplating strategy. Because TraI is also capable of sequencespecific DNA binding, this work paves the path to the final step of the biologically based strategy: addressable, targeted immobilization of nanoparticles on DNA. Inorganic binding peptides identified by several groups have demonstrated potential as molecular tools due to their high material affinity and specificity. Proteins non-specifically interact with gold, but our strategy relies on increased goldbinding specificity by including a gold-binding motif. By incorporating a gold-binding peptide while maintaining native DNA-binding function, we create a potent bifunctional molecular building block. The gold-binding peptide GBP1 (MHGKTQATSGTIQS), selected from a combinatorial peptide-display library, was incorporated into TraI in tandem repeats because previous research found that five (5 ) or seven (7 ) repeats of GBP1 exhibited a higher affinity for gold particles than a single copy. Se quences coding for the gold-binding peptides GBP1-5 , GBP17 , and the silica-binding control peptide QBP (LPDWWPPPQLYH) were inserted through molecular cloning techniques into the traI gene at a permissive site after codon 369 (Experimental Section); this permissive site lies in a region that codes for a large surface-exposed linker between the protein’s functional domains. The engineered TraI derivatives were tested for maintenance of their in vivo DNA-binding activity by comparison to wildtype TraI in a bacterial-mating assay. E. coli F’DtraI donor strains carrying plasmids with the complementing traI+, traI369GBP1-5 , traI369GBP1-7 , and traI369QBP alleles were proficient for F-plasmid transfer to a recipient strain, indicating that the essential DNA-binding activities (relaxase and helicase) of the encoded proteins were intact. The engineered TraI proteins were purified as previously described. Purified proteins exhibited DNA-dependent ATPase activity, indicating that these protein derivatives maintained their DNA-binding activity in vitro (Experimental Section). In our prior study, TraI with a cuprous oxide binding peptide (CN225) incorporated at a different permissive site near its C terminus was shown to precipitate cuprous oxide on DNA. However, the TraI inorganic binding derivatives of this study are the first to utilize internal permissive sites, as opposed to Nor C-terminal sites. Use of an internal permissive site circumvents the risk of degradation of inorganic binding repeats from the end of the protein and the specificity for gold instead of cuprous oxide provides an opportunity for electronic and photonic applications. Localized surface plasmon resonance (LSPR) analysis was used to measure the binding of the protein derivatives to AuNPs. Noble metal nanoparticles exhibit characteristic UV– visible absorption bands that result from their LSPR spectra. The peak extinction wavelength, lmax, of the LSPR spectrum [a] R. Hall Sedlak, E. Gachelet, L. Przybyla, Prof. B. Traxler Department of Microbiology, University of Washington Seattle, WA 98195 (USA) Fax: (+1)206-543-8297 E-mail : btraxler@u.washington.edu [b] M. Hnilova, Prof. M. Sarikaya, Prof. C. Tamerler Department of Materials Science and Engineering University of Washington Seattle, WA 98195 (USA) [c] D. Dranow, Prof. T. Gonen Department of Biochemistry, Howard Hughes Medical Institute University of Washington Seattle, WA 98195 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201000407.