Exploitation of peptide motif sequences and their use in nanobiotechnology

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Exploitation of peptide motif sequences and their use in nanobiotechnology Kiyotaka Shiba Short amino acid sequences extracted from natural proteins or created using in vitro evolution systems are sometimes associated with particular biological functions. These peptides, called peptide motifs, can serve as functional units for the creation of various tools for nanobiotechnology. In particular, peptide motifs that have the ability to specifically recognize the surfaces of solid materials and to mineralize certain inorganic materials have been linking biological science to material science. Here, I review how these peptide motifs have been isolated from natural proteins or created using in vitro evolution systems, and how they have been used in the nanobiotechnology field. Address Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Koto, Tokyo 135-8550, Japan Corresponding author: Shiba, Kiyotaka ([email protected])

Current Opinion in Biotechnology 2010, 21:412–425 This review comes from a themed issue on Nanobiotechnology Edited by Henry Hess and Luc Jaeger Available online 20th August 2010 0958-1669/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.07.008

Introduction Proteins and peptides are formed by the catenation of amino acids and can thus be thought of as a family of highmolecular-weight polymers. What distinguishes natural proteins/peptides from synthetic amino acid polymers is the complexity of their sequences. Natural proteins/peptides have complex sequences reflecting the combinatorics of amino acids. By contrast, the study of synthetic amino acid polymers has generally been limited to simple sequences such as poly-Asp and poly-Lys. While we are able to synthesize peptides with more complex sequences using the current technology, we do not yet know how to design complex sequences for particular purposes because it is unknown how particular aspects of sequence complexity will affect the functionality of the polymer. It follows that we have little ability to predict the tertiary structures of proteins/peptides from their sequences. Nevertheless, we do know that most natural proteins/ peptides efficiently fold into specific three-dimensional Current Opinion in Biotechnology 2010, 21:412–425

structures, which we believe to be a prerequisite for their functionality [1]. What biologists can do at this point is to predict the function of a given sequence based on its similarity to sequences of other proteins/peptides whose functions are already known. Although the sequence structure ! function relation remains a black box, the link between sequence and function can provide useful keys for synthesizing artificial macromolecules [2]. In other words, we can use peptide sequences as functional units, and the smaller the amino acid sequences are, the more flexible they can be for fabricating complex macromolecules. In this review, I will discuss how peptide motifs have been explored and used in nanobiotechnology.

Peptide motifs Once the association between a short amino acid sequence and its particular function is established, then that sequence is recognized as a peptide motif. Generally, there are two routes to the identification of peptide motifs (Figure 1). In the first, peptide motifs are extracted from the sequences of natural proteins; that is, the peptide motifs are defined as parts of natural proteins. There are several ways to define peptide motifs within a protein [2]. For instance, when the sequences of a given protein from various organisms are compared, we may notice that some portions within the sequences are well conserved. Similarly, comparisons of paralogous proteins that have evolved via gene duplication can lead to the identification of conserved sequences. Very often, the conservation of sequence indicates that the sequence is important. Indeed, in some cases, alteration of a conserved sequence results in the loss of function of the protein, which supports the linkage between sequence and function. These conserved sequences are then defined as peptide motifs. In addition, the sequences compared need not be limited to the same protein from different organisms or to paralogous groups. To the extent that informational analyses can reveal the nature of a sequence’s recurrence, they can provide clues for defining peptide motifs using combinations of genetic and/or biochemical analyses. In some cases, peptide motifs are scattered across a variety of proteins, while some peptide motifs recur within a single protein. Moreover, in some cases peptide motifs can be defined without sequence analysis. Minimalist approaches try to narrow down the cardinal portion from the whole structure by deleting unnecessary regions. This www.sciencedirect.com

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Figure 1

Natural and artificial peptide motifs. Natural peptide motifs are identified from natural protein sequences in different ways [2]. (a) Comparison of related sequences can reveal evolutionarily conserved regions (shown by red bars) in entire protein sequences (shown by gray bars), which are often defined as peptide motifs. (b) Repetitive sequences (shown by red bars) are often sources of peptide motif identification. (c) In a limited number of cases, biochemical analyses have identified peptide motifs without sequence comparison. (d) Artificial motifs are usually created as peptide binders (=aptamer) using in vitro evolution systems, among which the peptide-phage system is the most commonly used [19]. With that system, random peptide sequences are displayed on a filamentous phage particle. Phage clones that bind to a particular target molecule of interest are selected as binding peptides. Both natural and artificial motifs are concatamers of amino acids that have particular biological functions.

approach often results in the identification of a short peptide motif that retains the major function of the parental protein [2]. For the natural peptide motifs discussed above, the link between sequence and function is not always well understood. In these cases, a link can be proposed when a mutation within a peptide motif sequence leads to the loss of the protein’s function. To determine whether a peptide motif sequence is necessary and sufficient to exert its associated function, it is necessary to ask whether a synthetic version of the motif exerts the associated function, or the peptide must be shown to endow foreign molecules with the associated function through attachment to proteins (by genetic recombination) or to various nano-materials (by chemical modification). In addition to natural motifs, there are now a number of artificial peptide motifs that have been synthesized [2]. These peptide motifs are considered artificial motifs because the sequences are not necessarily found within the sequences of natural proteins, and instead of being the product of natural evolution they are obtained using artificial evolution systems first established in the early 1990s [3,4]. In these systems, artificial proteins/peptides (as well as RNA or DNA) are created (perhaps a better word would be ‘selected’) from pools of random www.sciencedirect.com

sequences based on the functionality of the molecules (Figure 1). The selected RNAs, DNAs or proteins/peptides are therefore associated with particular functions based on the nature of their creation process. If peptides are selected, they are regarded as peptide motifs. Such artificial evolution is the second route via which peptide motifs are defined. Experiments in which artificial motifs are created are further described in the section below.

Peptide motifs useful for nanobiotechnology What functions make peptide motifs useful tools for nanofabrication? Among the myriad functions allocated to natural proteins/peptides, some of those that have become a focus in nanobiotechnology are specific recognition, biomineralization [5–15,16,17,18], self-assembly, and self-organization. In the broadest sense of the expression, specific recognition is the foundation of all biological phenomena, while biomineralization, self-assembly, and self-organization reflect specific recognition between proteins/peptides and themselves or other molecules.

Peptide aptamers Peptide aptamers against inorganic materials

When we use the expression ‘specific recognition’ in the context of peptide motifs, we are referring to the selective binding of peptides to particular target molecules. These specifically binding peptides are often created using in Current Opinion in Biotechnology 2010, 21:412–425

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vitro evolution systems and are also called peptide aptamers. They are representative examples of artificial motifs.

Table 1

The phage-display peptide library system is a simple but robust tool for creating peptide binders of user-assigned targets, and it has been used to select peptides that bind to various receptors, antibodies and other biomolecules [19]. In the first decade after its establishment in 1990, the method was mainly used in the field of biology. In 2000, however, Belcher’s group first applied this system to select peptides with affinity for the surfaces of semiconductors [20]. They initially incubated a phage peptide library with a semiconductor substrate under aqueous conditions, after which they collected the phage that bound to the surface of the substrate and identified the sequence motifs that had affinity for semiconductors. Although the pioneering work of Brown (who employed a different evolutionary system) was published earlier [21], it was Belcher’s work that stimulated those who were interested in exploring the novel use of biomolecules in the emerging field of nanotechnology [7,8], and since then numerous peptide aptamers that bind to a variety of inorganic materials have been reported ([9,11], Table 1).

Metals Au Ag Pt Pd Pb Si Ti 6Al4VTi

[67,68,87] [23,38,88] [8] [8,89] [89] [23] [22,49,90] [91]

Metal oxides Al2O3 CoO Cr2O3 Cu2O Fe2O3 GeO2 IrO2 MnO2 PbO2 SiO2 TiO2 ZnO BaTiO3 Montmorillonite

[92,93] [94] [94] [95] [21,96] [97] [70] [94] [94] [98–100] [100,101] [95,102–104] [105,106] [45]

Compounds CdS CoPt FePt GaAs PbS ZnS

[107,108] [65,88,109] [65,110] [20] [107] [64,107]

Calcium compounds, etc. CaCO3 CaMoO4 Hydroxyapatite Zeolite

[111] [112] [113] [114]

Carbon materials Carbon black Carbon nanohorns Carbon nanotubes C60

[115] [116] [44,117–119] [120]

Polymers Cellulose PLLA PMMA PPyCl PS SPS

[121] [122] [123] [124] [125,126] [127]

Specificity and orthogonality of peptide aptamers

How specific are the aptamers created using in vitro evolution systems? In the standard protocol of a peptide-phage system, the selection experiment is performed within a plastic tube in the presence of only the target material, a blocking agent and some small chemicals (salts and pH-buffering chemicals). The experimental procedure is designed so that the selected phage binds to the target material but not to the blocking agent or plastic tube. Therefore, the guaranteed specificity is the ability to discriminate between the target material and the plastic tube, the blocking agent and certain chemical compounds. At this stage, whether the aptamer can distinguish the target material from other materials that were not included the selection process is not known. Similarly, whether the aptamer will retain its specificity in the absence of the blocking agent or under different solution conditions (pH, ionic strength, etc.) is unknown. Moreover, it is possible that the obtained peptide aptamer will only exhibit its binding specificity when it is displayed on a phage particle. In other words, the peptide could lose its binding ability when it is detached from the phage body. This aptamer evolved in a small world within a plastic tube and may only exert its selected function within the system where it evolved. Once the aptamer is put into a new environment, there is no guarantee that its specificity will be retained in this new system. As an illustrative example, TBP-1 (titanium-binding peptide) is a peptide aptamer that was isolated as a titanium Current Opinion in Biotechnology 2010, 21:412–425

Peptide aptamers against various inorganic materials Target material

Reference

binder using a peptide-phage system [22]. In that experiment, a phage library was incubated with titanium particles in the presence of 0.1% bovine serum albumin (a blocking agent), 50 mM Tris–HCl, pH 7.5 and 150 mM NaCl in a plastic tube. After washing out the unbound phage, the phage that bound titanium particles were detached by reducing the pH and were then propagated in bacteria. Although the actual selection process included several rounds of incubation (binding), washing, www.sciencedirect.com

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recovery, and propagation [19], the key to the strategy was to select peptides that bind to a titanium surface from among random sequences. At this stage, we knew that the selected phage could discriminate titanium particles from plastic and bovine serum albumin, but we did not know whether the phage had the capacity to bind other inorganic materials. We therefore next tested the phage’s ability to bind nine materials other than titanium, and we found that it did not bind gold, chrome, platinum, tin, zinc, copper or iron, but it did bind silver and silicon. On the basis of these results, it was concluded that the phage was a specific binder of titanium, silver, and silicon [23]. In other words, TBP-1 can distinguish titanium, silver, and silicon from gold, chrome, platinum, tin, zinc, copper, and iron. On the other hand, we cannot say whether TBP1 can distinguish titanium from an untested material such as aluminum. Our knowledge of the surface properties of inorganic materials under aqueous conditions is still very limited [24,25]. Even if we knew the characteristics of the surfaces of materials on an atomic scale, we would not know how peptide aptamers selectively recognize these characteristics and interact with the surface. Consequently, whether or not the peptide will bind to aluminum must be determined experimentally. We often say ‘This peptide is a specific ligand for receptor X’ or ‘This antibody is highly specific.’ In these everyday terms, we implicitly define the environment or system in which a peptide ligand or antibody should act in a specific manner. When a peptide ligand specifically stimulates a certain signal transduction pathway in a mouse, or when an antibody detects its target protein in an immunoblotting experiment using cell extract from a particular human cell line, the molecules are deemed to show satisfactory specificity. Nonetheless, we do not know whether the peptide ligand would act on additional signaling pathways in humans (but not mice) or whether the antibody would cross-react with other proteins extracted from organs (but not the cell line). Thus, specific interactions can only be assessed when the system that surrounds the interacting molecules is well defined because a specific interaction observed in system A may not occur in system B (Figure 2). When a specific interaction between molecule-1 and molecule-2 in system A also occurs in system B, we say that the interaction has ‘orthogonality’ in the two systems. It is important to pay close attention to the orthogonality of interactions, especially when the interaction was created using an in vitro evolution system. Because the specific interaction of an aptamer and its target molecule is selected in a simple environment consisting of only a plastic tube and few chemical compounds, whether the evolved interaction will retain its orthogonality in a new environment is unknown. When orthogonality is taken into account, it becomes apparent that the selection should be carried out within an environwww.sciencedirect.com

Figure 2

Orthogonality of specificity. The specificity of a molecule can only be discussed when the system in which the molecule exists is defined. In vitro evolution systems create artificial molecules that specifically function within that system. However, this orthogonality does not necessarily persist when the molecule is transferred to a new system. Researchers must take orthogonality (=specificity in a given system) into consideration and carefully design the evolution conditions (‘System A’ in the figure) so that it retains its orthogonality in the end-point system (‘System B’ in the figure). In the figure, the evolved artificial molecule is represented by the brown crescent (molecule-1) and its acting target is shown by the green circle (molecule-2). The other circles represent components present in the end-point system (System B). Researchers try to recapitulate the end-point system in their evolution system (System A). However, the evolution system inevitably includes components that are not included in ‘System B’ (denoted by stars). In addition, it is generally very difficult to have a complete picture of a natural system (System B), and therefore its recapitulation has limits.

ment similar to that in which the selected molecule will be utilized. For instance, if one wanted to create a peptide aptamer that can deliver anticancer drugs to tumors after circulating in the bloodstream, one might want to select the peptide aptamer in the body (in vivo selection) (Figure 3). Similar attention to the orthogonality of peptide specificity is also important when peptides are employed in nanotechnology. The environments in which peptide motifs play roles in nanotechnology are generally composed of inorganic materials such as silicon and semiconductors. If the environment in which the peptide motif should be active is known, the in vitro evolution conditions can be set such that they will endow the peptide with the fullest orthogonality during the selection process. In the case of TBP-1, our first aim was to functioCurrent Opinion in Biotechnology 2010, 21:412–425

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Figure 3

Orthogonality-conscious in vitro evolution experiments. If the end-point environment in which an evolved motif will be used is known, the selection conditions can be set so that they are close to the end-point environment. (a) For instance, if a peptide aptamer is to be used to deliver a drug-loaded carrier to a tumor via the bloodstream, a peptide-phage library can be intravenously injected into a mouse, and phage clones that accumulate in the region of the tumor can then be selected [84]. In the illustration, a biomarker molecule on a tumor is represented as a green circle, while the other circles show components of a biological network. The brown crescent denotes a peptide aptamer. (b) If aptamers are to be used for nanofabricating electronic devices, the selection conditions should be designed so that the evolved aptamer retains its orthogonality in that final environment. In the illustration, a target inorganic material is represented as a green star while the stars of other colors indicate materials that will be used in the final device (but that should not be a target of the peptide aptamer). Circles show components included in the in vitro evolution system (such as phage proteins and bacterial proteins). The brown crescent denotes a peptide aptamer.

nalize the surface of medical titanium materials [26,27], and we did not pay much attention to the peptide’s ability to discriminate titanium from other inorganic materials. Only later, when we began to use TBP-1 in the fabrication of nanodevices, did its ability to discriminate between inorganic materials come into play. Because TBP-1 is able to discriminate titanium from platinum, we were able to selectively allocate TBP-1-ornamented nanomaterials onto nanometric titanium patterns deposited on a platinum substrate [28,29]. However, the affinity of TBP1 for silicon and silicon dioxide was not favorable for nanofabrication, as many devices are fabricated on a silicon or silicon dioxide substrate. We circumvented this issue by seeking solution conditions that would enable TBP-1 to distinguish between titanium and silicon dioxide; that is, conditions that assure the orthogonality of the peptide in a system composed of titanium and silicon dioxide [30]. Under these newly established conditions, we were able to selectively target TBP-1 to titanium regions deposited on silicon dioxide. Thus, in the case of TBP-1, serendipity contributed to the establishment of orthogonality in an application for device fabrication. If our research had started with the aim of evolving peptide aptamers to be used for selective Current Opinion in Biotechnology 2010, 21:412–425

positioning with titanium on silicon dioxide, we would have been able to set the conditions so as to evolve a peptide that would distinguish these materials under the given conditions. Namely, a peptide would be first counter-selected against silicon dioxide before being selected against titanium (Figure 3). In vitro selection reliably allows the isolation of biomolecules adapted to very specific environments. Consequently, careful design of the selection conditions will lead to the creation of peptide aptamers with orthogonality in the end-point environment. Mineralizing activities of peptide motifs

Biomineralization has attracted attention in the field of bio-nanotechnology because firstly, biomineralization reactions proceed under milder conditions than conventional industrial methods and secondly, the hard tissues obtained through biomineralization often have elaborately designed nanostructures. These features of biomineralization suggest that novel methods for nanofabrication will be developed in which elaborately designed crystals of inorganic material are fabricated under environmentally benign conditions, that is, conditions that do not require high temperature, pressure or energy. www.sciencedirect.com

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Although the phenomenon of biomineralization has captivated scientists’ curiosity for centuries [31], our knowledge of the mechanisms by which biomolecules accelerate crystallization processes, and how biomolecules determine the appearance and characteristics of crystals, remains very limited. It has been proposed that the biomineralization mechanism involves the use of structural complementarity between the surface of a crystal and a biomolecule that (in some way) modulates crystal growth. This idea was first proposed by Weiner and Hood after they noticed repetitive structures in proteins isolated from mollusk shells [32]. The proteins contained repeats of either Asp-Gly or Asp-Ser, and they speculated that the regularly spaced aspartic acid side chains function as a template upon which mineralization of calcium carbonate occurs. It has also been proposed that similar structural regularity is responsible for the suppression of ice formation by some antifreeze proteins [33,34]. In that case, regularly spaced threonine side chains complement the crystal lattice of ice. Although further studies will be needed before one can definitively state that the periodic structures of proteins mediate the recognition of periodicities within crystals, researchers have become interested in the mineralization ability of biomolecules whose structures are complementary to the surfaces of crystals [35,36]; in other words, biomolecules that specifically recognize the surfaces of inorganic materials. Indeed, many aptamers that bind inorganic materials are reportedly able to mediate mineralization of their target atoms [23,37–39]. For instance, the peptide aptamer that was created as a binder to silver has the ability to accelerate the growth of silver nanoparticles, which enabled its use in the fabrication of arrays of silver crystals on a glass substrate [38]. Several sequences within natural proteins have been identified as mineralizing motifs [40–43]. However, because living systems mineralize only a limited set of inorganic materials (calcium phosphate, calcium carbonate, silica and ferrihydrite, among others), these natural motifs should not be expected to mediate the mineralization of materials that are important to industry (e.g. semiconductors). On the other hand, by using an in vitro evolution system, binders of materials that living systems have not encountered during their biological evolution can be created artificially, and mineralization activity can be drawn from peptide aptamers, which will greatly extend the repertoire of biomineralization. Motif-programming for nanobiotechnology

Because peptide motifs serve as functional units, multifunctionality can often be achieved by combining peptide motifs. For instance, Naik’s group has isolated a carbon nanotube-binding peptide, P1, and conjugated it to a natural silicification peptide motif, R5. The resultant 35-mer peptide is bifunctional that is it binds to the surface of carbon nanotubes and endows them with www.sciencedirect.com

silicification activity [44]. Using a similar approach, they also succeeded at assembling metal nanoparticles on montmorillonite by conjugating peptide aptamers against montmorillonite and metals [45]. Recently, the group expanded this surface modification experiment by fusing P1 peptide with a peptide derived from the binding pocket of the honeybee odor binding protein ASP1, which is known to bind to trinitrotoluene (TNT) [46]. Although their research is still in the initial stages, their goal is to develop a novel TNT sensor based on a carbonnanotube field effect transistor, which should be applicable to other biomimetic chemosensors by changing the sensing peptide motif unit. Titanium and certain other inorganic materials are now commonly used in medical devices such as artificial joints and dental implants. The functionalization of the surfaces of these medical devices using motif-programming has been proposed by several groups. For instance, Sarikaya’s group made a conjugate between a titanium-binding peptide and a natural cell attachment motif (RGD) to functionalize a titanium implant [47]. Similarly, Yoshinari’s group has synthesized a conjugated peptide between TBP-1 and an antimicrobial peptide for the prevention of biofilm formation on titanium implants [48]. Above are examples of motif-programming using two peptide motifs. Bifunctional molecules can also be created by conjugating peptide motifs with high molecular weight polymer compounds. Polyethylene glycol (PEG) is a widely used amphipathic polymer with excellent biocompatibility. The conjugation of PEG with a peptide aptamer against a certain medical material is expected to enhance the biocompatibility of the material. Examples of this approach include the conjugation of a titaniumbinding peptide [49] with PEG as well as that of a carbon nanohorn binding peptide with PEG [50,51]. Utilization of bifunctional peptide aptamers in nanofabrication

As mentioned, TBP-1 is an artificial motif with the ability to bind titanium, silicon and silver [22,23]. This peptide also has the ability to enhance the mineralization of these materials [23,29]. TBP-1 is thus bifunctional: it is both a binder and a modulator of mineralization. We endowed ferritin (a spherical protein composed of 24 subunits [52] and capable of holding various nano-dots within its inner space [53–55]) with the bifunctionality of TBP-1 by fusing the core sequence of TBP-1 to the N-terminal end of the ferritin subunit [56]. The engineered ferritin, minT1-LF, exhibited selective binding to titanium [28,30,57–59] and an enhanced capacity for mediating silica [60] and titania [29] formation. By taking advantage of the bifunctionality of minT1-LF, we developed a novel technique for nanofabrication, which we named BioLBL [29,60,61] (Figure 4). BioLBL starts with the Current Opinion in Biotechnology 2010, 21:412–425

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Figure 4

Formation of a multilayer construction using the binding and mineralization activities of a peptide motif (BioLBL) [29,60,61]. (a) minT1-LF is an engineered ferritin derived from TBP-1 [22] endowed with titanium-binding and titania-forming abilities [56]. (b) First, minT1-LF is positioned on titanium regions patterned on platinum film. (c) On this first ferritin layer, the first intercalating layer of titania is formed. (d) This titania layer then serves as a binding target for a second minT1-LF layer. By repeating these binding and mineralization cycles, three-dimensional multilayer structures are built up on the substrate. (e) The image shows a field emission type scanning electron micrograph of a cleaved site of the substrate containing six layers of minT1-LF and titania [29].

specific binding of minT1-LF to titanium nano-patterned on an appropriate substrate such as platinum. Within minT1-LF, 24 peptide aptamers are nearly symmetrically distributed on the surface of each ferritin molecule, some of which are free to access chemical compounds in solution. Consequently, upon addition of prehydrolyzed Ti(IV) bis(ammonium lactato)-dihydroxide, a thin titania film forms on the minT1-LF layer. The newly formed titania layer then serves as a binding target for minT1-LF, and a second minT1-LF monolayer is formed on the titania layer. This cycle of minT1-LF binding and mineralization can be repeated until the desired number of layers is reached. Because each ferritin layer is separated by a thin titania layer (silica layers can also be used), interlayer diffusion (which is often problematic with conventional layer-by-layer construction methods [62]) of minT1-LF is prevented, and each ferritin layer is well separated from all others, even in the vicinity of the base substrate. Moreover, the excellent ability of BioLBL to segregate one layer from the others enabled us to fabricate heterogeneous multilayer structures through the use of different metal-containing ferritins [60]. Current Opinion in Biotechnology 2010, 21:412–425

Epigenetic processing and secondary evolution of peptide motifs Genetical and chemical modifications

In the preceding section, I noted that the specificity of a peptide aptamer can only be discussed when the system in which the aptamer functions is defined. The environment in which the evolved aptamer will be used must be considered, and the conditions for the in vitro evolution should be as close to the final environment as possible (Figure 3). For example, if one wants to use a peptide aptamer within the circulatory system, one should select that aptamer using an in vivo selection system. But if a peptide is needed for fabricating an electronic device, the peptide should be selected in a system composed of inorganic materials. However, one inevitable limitation of the current methodologies is that peptide aptamers must be selected in a form that is linked to a phage particle [19] or some other genetic module [63] (Figure 1). Thus, the orthogonality of the peptide is only assured when it is displayed on its phage particle (or other display module), and whether a peptide aptamer will retain its orthogonality when it is detached from the www.sciencedirect.com

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phage cannot be predicted. In some cases, therefore, the most effective way of applying peptide aptamers is to use the entire peptide-displaying phage. The most commonly used phage in peptide-phage systems is a filamentous phage that has a high aspect ratio and behaves like a highmolecular-weight polymer [19]. By taking advantage of the unique properties of this aptamer-displaying filamentous phage, Belcher’s group was able to develop novel types of electronic tools [64–66]. In a study published on 2006, they engineered a filamentous phage so that it could display two distinct peptide motifs on its surface. One peptide motif was a tetraglutamate sequence, E4, which

was rationally designed so that it could serve as a cobalt ion nucleating motif. The second peptide motif was a dodecapeptide gold binding motif that they isolated using a phage-display peptide library system (Table 1). Using this engineered phage, (AuE4), they first attached 5-nm gold nanoparticles on its surface by virtue of the displayed gold binding motif, and then they grew Co3O4 via the tetraglutamate functionality. The obtained Au–Co3O4 hybrid nanowire showed excellent electrochemical properties, and it could be potentially used as an electrode for advanced lithium batteries [67]. Recently, using a phage displaying both a tetraglutamate motif and a peptide

Figure 5

Processing peptide motifs to enhance their lateral functionality. (a) Chemical modifications (shown in green) are sometime able to enhance peptide motif (shown in red) functionality. (b) Conjugation of peptide motifs (shown in red box) with foreign molecules (such as polyethylene glycol) can be used to facilitate clinical application of the motif [50,51]. (c) Recombinant methodologies are used to endow natural proteins with peptide motif functions. In this example, a DNA cassette that encodes the minTBP-1 peptide motif was inserted at the 50 end of the gene for a ferritin subunit so that minTBP-1 and the ferritin subunit are translated as a single fusion protein. The fusion protein assembled into a ferritin-like particle [56] on which minTBP-1 (shown in red) was displayed [28]. (d) In MolCraft, combinatorial polymers are prepared from more than two peptide motifs (shown in red and blue), from which clones showing the functionality of the embedded motifs are selected [26,27,72,74,75,79–81,128]. www.sciencedirect.com

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Figure 6

aptamer against gold (different from the one used in the above experiment, Table 1 [68]), they have succeeded at synthesizing a porous nanowire network composed of AuAg alloy, which should improve the retentive capacity of lithium ion batteries [69]. The group also assembled photocatalytic nanostructures on a phage displaying a peptide aptamer against iridium oxide (IrO2) [70] (Table 1), expanding the possibilities for the use of engineered phage in nanobiotechnology. In most cases, however, peptide aptamers are used in their detached form (synthetic peptides) or in a reattached form (recombinants or conjugates), the properties of which may differ from those of the phage-attached aptamer. For instance, TBP-1 strongly binds titanium when displayed on its phage, but the detached TBP-1 synthetic peptide shows only weak binding that is not sufficient for various applications [23]. Notably, its strong binding is restored when TBP-1 is reattached to ferritin [56] or a chemical nano-cage [71]. However, only modest binding is restored when TBP-1 is reattached to certain other molecules [27]. Thus, the function of a peptide motif is greatly influenced by the molecule that presents the peptide motif and the means by which the peptide motif is presented by the molecule, which brings to mind the orthogonality of peptide specificity discussed in the preceding section. Because the molecular context that surrounds a peptide motif can be regarded as a microscopic system, here again the function of a peptide motif does not necessarily persist when it is transferred to a different system. Context similarly influences the activities of natural motifs [72], which often lose their associated functions when they are transferred to foreign molecules [73–76]. To maximize the potential function of natural and artificial motifs within a new context, trial and error can be used to search for solutions that restore the original functions of peptide motifs (Figure 5). Epigenetic processing — that is, chemical modification — of peptide motifs is one approach. It has been shown, for example,

Motif-programming using MolCraft [2,79]. (a) With MolCraft, a single microgene is first designed so that different peptide motifs are encoded by different reading frames of the DNA sequence. In the example, three peptide motifs were embedded in a gene of 68 base pairs (the microgene MG-69). The red box shows the minTBP-1 motif, which has ability to bind the surface of titanium [22]. The blue and green boxes represent natural motifs derived from a dentin matrix protein, which have been reported to enhance mineralization of calcium phosphate when immobilized on a glass substrate [42]. (b) On the basis of the sequence of a designer microgene, sense and antisense MPR primers (indicated as MPR primer-1 and 2, respectively) are synthesized. MPR primer-1 and 2 share 30 sequences that enable base-pair formation between the primers, but contain mismatched base pairs at their 30 -OH ends (shown by red letters with dots). (c) The thermal cycle reaction with these MPR primers and a thermostable DNA polymerase having 30 –50 exonuclease activity results in the formation of tandem repeats of the microgene

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[129]. This polymerization reaction (microgene polymerization reaction, MPR) proceeds efficiently when MPR primers have mismatched base pairs at their 30 -OH ends. MPR introduces random insertions and deletion mutations into the junctions of the microgene units, making the polymer a combinatorial library of three reading frames. (d) The microgene polymers are cloned into an expression vector and one of the reading frames is translated in bacterial or mammalian cells. The resulting protein products contain three motifs embedded in the microgene in various orders and in various numbers. The schematic structures of some of the artificial proteins are shown, in which the color coding is the same as in (a). (e) From the protein library, the clones that have the expected functions are selected. In this example [130], proteins that can endow the surface of a titanium substrate with calcification ability were selected using an in vitro calcification assay. The left photograph shows titanium plates after incubation with protein and calcium and phosphate ions. An electron microscopic image of the resulting calcium phosphate crystals is shown on the right.

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that a latent motif function can be made manifest by a chemical modification that endows the peptide with alpha helicity [77], and that conjugation of peptide motifs with foreign molecules (such as high-molecular-weight chemical polymers) can be applied to achieve practical goals. For instance, it has been shown that the conjugation of PEG to a peptide targeting tumor cells improves the peptide’s ability to image tumors in vivo via positron emission tomography (PET) [78]. Motif-programming by MolCraft

MolCraft is a system that was developed for motif-based artificial protein construction [79] (Figure 5). In the preceding section, I introduced motif-programming experiments in which multifunctional peptides are created by conjugating more than two peptide motifs. However, simple conjugation does not always work for motifprogramming. In other words, simple arithmetic addition does not always work for peptide motifs. When difficulties are encountered in motif-programming via simple conjugation, the MolCraft system [75,80] can be employed. In MolCraft, a protein library that contains different motifs in various orders and numbers is first constructed, from which clones showing the functionality of the embedded motifs are selected. For instance, when wanted to create an artificial protein able to crystallize calcium phosphate on the surface of a titanium implant (which could improve the biocompatibility of the titanium implant), we first designed a microgene embedded with a titanium-binding motif and a calcification motif, and then we made a protein library by polymerizing the microgene [27,76]. From the library, proteins that enhance the in vitro crystallization of calcium phosphate on the titanium substrate were selected (Figure 6). MolCraft is therefore regarded as a hierarchical evolution method: starting from peptide motifs, larger proteins with higher functions are selected. In addition to the abovementioned minimization proteins, using the MolCraft system we have created artificial cytokines from natural signal transduction related motifs [73,74,80,81], artificial matrix proteins for titanium implants from minTBP-1 and cell binding motifs [26] and an artificial signaling domain from Srk phosphorylation motifs [82].

revolutionize nanobiotechnological research. That said, the lack of robustness of peptide motifs when they are removed from their parental protein remains a limitation of this approach. Although natural motifs can recapitulate their parental functions under certain conditions, that functionality is often lost when environmental conditions change. Similarly, artificial motifs faithfully express their functions within the systems where they evolved, but they do not always retain their original functions in different systems. The acquisition of robustness by living systems over the course of evolution is an important theme of basic biological science [83]. The current in vitro evolution systems and decompositional motif-based research may lack one or more critical elements that contribute to the robustness of biological systems. Nevertheless, the trial and error approach to synthesizing novel molecules from peptide motifs should deepen our understanding of the basis of robustness of biological systems, which is woven from the combinatorics of 20 amino acids.

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127. Serizawa T, Techawanitchai P, Matsuno H: Isolation of peptides that can recognize syndiotactic polystyrene. Chembiochem 2007, 8:989-993. 128. Chirila TV, Minamisawa T, Keen I, Shiba K: Effect of motifprogrammed artificial proteins on the calcium uptake in a synthetic hydrogel. Macromol Biosci 2009, 9:959-967. 129. Shiba K, Takahashi T, Noda T: Creation of libraries with long open reading frames by polymerization of a microgene. Proc Natl Acad Sci U S A 1997, 94:3805-3810. 130. Tsuji T, Oaki Y, Yoshinari M, Kato T, Shiba K: Motif-programmed artificial proteins mediated nucleation of octacalcium phosphate on the titanium substrates. Chem Comm 2010, in press, doi:10.1039/C0CC01512A. Glossary Aptamer [3]: A biomolecule with specific binding capacity created using an in vitro evolution system. The term was coined by Ellington and Szostak when they selected RNA molecules that bound to dye molecules (which they assigned as target molecules) starting from a pool of artificial RNAs with random sequences. Biomineralization [31]: The process by which living organisms form hard solid structures from inorganic compounds. Our bones and teeth, the nacreous layers of pearls, sea urchin spines and diatom frustules are all products of biomineralization. In vivo selection [84–86]: Many organ-specific and tumor-specific peptide aptamers have been created by intravenously injecting mice with a peptide-phage library. In this kind of in vivo selection, the orthogonality

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of the interaction between the peptide and its target is assured in mice, but the interaction might not always be orthogonal in the human body. If the end-point of the aptamer search is a clinical application, the ideal setting for the selection would be the human body. Currently, ex vivo selection procedures using surgically isolated tumors have been used to produce peptide aptamers that retain satisfactory orthogonality in clinical situations. MolCraft [79]: An in vitro protein evolution system in which a microgene is initially designed so that motifs are encoded by different reading frames, and the microgene is then tandemly polymerized with insertion or deletion mutations at the junctions between microgene units. Because of the junctional perturbations, the proteins translated from a single microgene polymer are molecularly diverse (as they originate from the combinatorics of three reading frames) and they are thus combinatorial polymers of three peptide motifs. Paralogous (adj.): Genes (or proteins) that are believed to have evolved by gene duplication are designated ‘paralogous genes.’ Paralogous genes generally share sequence similarities. Peptide motifs: Short amino acid sequences that have been associated with particular biological functions. Phage-display peptide library system [4,19]: An in vitro evolution system in which random peptide sequences are displayed on phage particles. Because the peptide sequences are encoded by DNA fragments inserted into the phage genome, the peptide sequence can be deduced by sequencing the DNA after the binding phage are selected.

Current Opinion in Biotechnology 2010, 21:412–425