We report a virus-based scaffold for the synthesis of single-crystal ZnS, CdS, and freestanding chemically ordered CoPt and FePt nanowires, with the means of modifying substrate specificity through standard biological methods. Peptides (selected through an evolutionary screening process) that exhibit control of composition, size, and phase during nanoparticle nucleation have been expressed on the highly ordered filamentous capsid of the M13 bacteriophage. The incorporation of specific, nucleating peptides into the generic scaffold of the M13 coat structure provides a viable template for the directed synthesis of semiconducting and magnetic materials. Removal of the viral template by means of annealing promoted oriented aggregation-based crystal growth, forming individual crystalline nanowires. The unique ability to interchange substrate-specific peptides into the linear self-assembled filamentous construct of the M13 virus introduces a material tunability that has not been seen in previous synthetic routes. Therefore, this system provides a genetic toolkit for growing and organizing nanowires from semiconducting and magnetic materials.

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure. E xploiting the biologically evolved, self-assembling structures of viral capsids is a possible mechanism in the ordering of technologically important materials. A key characteristic of materials synthesized by natural biological systems is the hierarchical organization of structures on many length scales with controlled size, shape, alignment, and orientation (1-8). There has been much interest in using biological templates in vitro to modulate the growth of inorganic, semiconductor, magnetic, and other technologically important materials (9-14). Our previous work, along with that of others, has shown that biological combinatorial approaches can identify amino acid sequences capable of interacting with a variety of materials (11,15). Here, we present a system that exploits the self-assembling, crystalline structure of the viral coat to nucleate and template II-VI semiconductor nanocrystals into highly oriented quantum dot nanowires. To accomplish this, the pVIII major coat protein was engineered to display evolutionary-selected peptides, enabling directed nanocrystal growth into viral-semiconductor hybrid nanowires that were crystallographically oriented over the micrometer size range.The helical major coat protein, pVIII, of viruses can be engineered to express fusion proteins that readily self-assemble into a highly oriented viral coat structure. A 5-fold axis of rotation relates symmetric points on the coat (16, 17). Peptide sequences engineered into the pVIII protein for use as nanocrystal templates were select...

Semiconductor nanocrystals, which have unique optical and electronic properties, have potential for applications in the emerging field of nanoelectronics. To produce nanocrystals cheaply and efficiently, biological methods of synthesis are being explored. We found that E. coli, when incubated with cadmium chloride and sodium sulfide, have the capacity to synthesize intracellular cadmium sulfide (CdS) nanocrystals. The nanocrystals are composed of a wurtzite crystal phase with a size distribution of 2-5 nm. Nanocrystal biosynthesis increased about 20-fold in E. coli cells grown to stationary phase compared to late logarithmic phase. Our results highlight how different genetic and physiological parameters can enhance the formation of nanocrystals within bacterial cells.

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One barrier to the construction of nanoscale devices is the ability to place materials into 2D- and 3D-ordered arrays by controlling the assembly and ordering of connections between nanomaterials. Ordered assembly of nanoscale materials may potentially be achieved using biological tools that direct specific connections between individual components. Recently, viruses were successfully employed as scaffolds for the nucleation of nanoparticles and nanowires (Mao et al., 2004); however, there is a paucity of methods for the higher order assembly of phage-templated materials. Here we describe a general strategy for the assembly of filamentous bacteriophages into long, wire-like or into tripod-like structures. To prepare the linear phage assemblies, dimeric leucine zipper protein domains, fused to the p3 and p9 proteins of M13 bacteriophage, were employed to direct the specific end-to-end self-association of the bacteriophage particles. Electron microscopy revealed that up to 90% of the phage displaying complementary leucine zipper domains formed linear multi-phage assemblies, composed of up to 30 phage in length. To prepare tripod-like assemblies, phage were engineered to express trimeric leucine zippers as p3 fusion proteins. This resulted in 3D assembly with three individual phages attached at a single point. These ordered phage structures should provide a foundation for self-assembly of virally templated nanomaterials into useful devices.

Ribonuclease A (RNase A) catalyzes the cleavage of RNA after pyrimidine nucleotides. When bound in the active site, the base of a pyrimidine nucleotide forms hydrogen bonds with the side chain of Thr45. Here, the role of Thr45 was probed by using the wild-type enzyme, its T45G variant, X-ray diffraction analysis, and synthetic oligonucleotides as ligands and substrates. Catalytic specificity was determined with the fluorogenic substrate: 6-carboxyfluorescein approximately dArXdAdA approximately 6-carboxytetramethylrhodamine (6-FAM approximately dArXdAdA approximately 6-TAMRA), where X = C, U, A, or G. Wild-type RNase A cleaves 10(6)-fold faster when X = C than when X = A. Likewise, its affinity for the non-hydrolyzable oligonucleotide 6-FAM approximately d(CAA) is 50-fold greater than for 6-FAM approximately d(AAA). T45G RNase A cleaves 6-FAM approximately dArAdAdA approximately 6-TAMRA 10(2)-fold faster than does the wild-type enzyme. The structure of crystalline T45G RNase A, determined at 1.8-A resolution by X-ray diffraction analysis, does not reveal new potential interactions with a nucleobase. Indeed, the two enzymes have a similar affinity for 6-FAM approximately d(AAA). The importance of pentofuranosyl ring conformation to nucleotide specificity was probed with 6-FAM approximately d(AU(F)AA), where U(F) is 2'-deoxy-2'-fluorouridine. The conformation of the pentofuranosyl ring in dU(F) is known to be more similar to that in rU than dU. The affinity of wild-type RNase A for 6-FAM approximately d(AU(F)AA) is 50-fold lower than for 6-FAM approximately d(AUAA). This discrimination is lost in the T45G enzyme. Together, these data indicate that the side chain of Thr45 plays multiple roles-interacting favorably with pyrimidine nucleobases but unfavorably with purine nucleobases. Moreover, a ribose-like ring disfavors the interaction of Thr45 with a pyrimidine nucleobase, suggesting that Thr45 enhances catalysis by ground-state destabilization.

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2′‐Fluoro‐2′‐deoxyuridine 3′‐phosphate (dUFMP) and arabinouridine 3′‐phosphate (araUMP) have non‐natural furanose rings. dUFMP and araUMP were prepared by chemical synthesis and found to have three‐ to sevenfold higher affinity than uridine 3′‐phosphate (3′‐UMP) or 2′‐deoxyuridine 3′‐phosphate (dUMP) for ribonuclease A (RNase A). These differences probably arise (in part) from the phosphoryl groups of 3′‐UMP, dUFMP, and araUMP (pKa = 5.9) being more anionic than that of dUMP (pKa = 6.3). The three‐dimensional structures of the crystalline complexes of RNase A with dUMP, dUFMP and araUMP were determined at < 1.7 Å resolution by X‐ray diffraction analysis. In these three structures, the uracil nucleobases and phosphoryl groups bind to the enzyme in a nearly identical position. Unlike 3′‐UMP and dUFMP, dUMP and araUMP bind with their furanose rings in the preferred pucker. In the RNase A·araUMP complex, the 2′‐hydroxyl group is exposed to the solvent. All four 3′‐nucleotides bind more tightly to wild‐type RNase A than to its T45G variant, which lacks the residue that interacts most closely with the uracil nucleobase. These findings illuminate in atomic detail the interaction of RNase A and 3′‐nucleotides, and indicate that non‐natural furanose rings can serve as the basis for more potent inhibitors of catalysis by RNase A.