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Notes Affinity Selection of Peptide Phage Libraries against Single-Wall Carbon Nanohorns Identifies a Peptide Aptamer with Conformational Variability Daisuke Kase,† John L. Kulp, III,‡ Masako Yudasaka,§ John Spencer Evans,‡ Sumio Iijima,§,| and Kiyotaka Shiba*,†,⊥ Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Toshima, Tokyo 170-8455, Japan, Laboratory for Chemical Physics, New York University, New York, New York 10010, NEC, Miyukigaoka, Tsukuba 305-8501, Japan, SORST, JST, c/o NEC, Meijo University, Tenpaku, Nagoya 468-8502, Japan, AIST, Higashi, Tsukuba 305-8565, Japan, and CREST, JST, c/o Cancer Institute Received April 23, 2004. In Final Form: July 19, 2004
Introduction Single-wall carbon nanohorns (SWNHs) represent recently discovered carbonaceous nanostructured materials.1 They are spherical aggregates of elongated graphitic tubes, and these aggregates possess a homogeneous diameter distribution around 80-100 nm.1 Each individual tube is ∼2-3 nm in diameter and 50 nm in length. The individual tubes feature closed ends with corn-shaped caps that permit assembly into “dahlia-like” or “bud-like” spherical nanostructures, depending upon the synthesis conditions.2 SWNHs are produced in large quantities (1050 g/h) by laser ablation of graphite, a process which does not require a metal catalyst and thus enables preparation with unparalleled purity (>90%).1 Along with their large surface area and molecular sieving effect,3 SWNHs have come under close scrutiny for possible application in the field of molecular adsorption.4,5 The unique physicochemical properties of SWNHs also suggest their possible inclusion within the medical field for applications ranging from drug delivery systems (DDS) to noninvasive imaging. However, for the successful application of SWNHs in nanomedicine, it would be desirable that their overall function be enhanced via †
Japanese Foundation for Cancer Research. New York University. § NEC and SORST, JST, c/o NEC. | Meijo University and AIST. ⊥ CREST, JST, c/o Cancer Institute. ‡
(1) (a) Iijima, S.; Yudasaka, M.; Yamada, R.; Bandow, S.; Suenaga, K.; Kokai, F.; Takahashi, K. Chem. Phys. Lett. 1999, 309, 165. (b) Iijima, S. Physica B 2002, 323, 1. (2) Kasuya, D.; Yudasaka, M.; Takahashi, K.; Kokai, F.; Iijima, S. J. Phys. Chem. B 2002, 106, 4947. (3) Murata, K.; Hirahara, K.; Yudasaka, M.; Iijima, S.; Kasuya, D.; Kaneko, K. J. Phys. Chem. B 2002, 106, 12668. (4) Nisha, J. A.; Yudasaka, M.; Bandow, S.; Kokai, F.; Takahashi, K.; Iijima, S. Chem. Phys. Lett. 2000, 328, 381. (5) (a) Bekyarova, E.; Kaneko, K.; Kasuya, D.; Takahashi, K.; Kokai, F.; Yudasaka, M.; Iijima, S. Physica B 2002, 323, 143. (b) Ohba, T.; Murata, K.; Kaneko, K.; Steele, W. A.; Kokai, F.; Takahashi, K.; Kasuya, D.; Yudasaka, M.; Iijima, S. Nano Lett. 2001, 1, 371. (c) Yoshitake, T.; Shimakawa, Y.; Kuroshima, S.; Kimura, H.; Ichihashi, T.; Kubo, Y.; Kasuya, D.; Takahashi, K.; Kokai, F.; Yudasaka, M.; Iijima, S. Physica B 2002, 323, 124.
coupling with biomolecules having various biological functions. For example, organ specificities, organelle targeting, and molecular recognition are among the SWNH functions that could be generated via the use of specific proteins or individual sequence motifs coupled to exposed nanotube surfaces. With the long-term goal of developing novel nanocomposites consisting of SWNHs and artificial proteins, we report here the use of peptide phage display methodology6 to select peptide motifs that specifically recognize the exposed surfaces of SWNHs. Experimental Section The “dahlia-like” SWNHs were prepared by CO2 laser ablation under an Ar gas atmosphere (6 × 104 Pa) at room temperature.1 To apply the technique of peptide phage display and screening to the SWNHs, we needed to explore the use of immobilized SWNHs due to the fluffy nature of the aggregates and the difficulties associated with the separation of bound phages from unbound phages from these materials. Therefore, prior to phage library screening, we pretreated SWNHs with 70% HNO3 at 400 K for 1 h to generate COOH groups on their surfaces.7 The biotinylated SWNHs were then immobilized streptavidin-coated magnetic beads (M-280, Dynal, Oslo). The SWNH magnetic bead complexes are easily recovered using a magnet apparatus, thus enabling us to subsequently perform a standard affinity selection by phage panning against the immobilized SWNHs (Supporting Information). For competition assays with synthetic peptides, we immobilized the biotinylated SWNHs to a plastic plate (Immulon 4HBH, Dynex, Chantilly) which was first coated with streptavidin (New England Biolabs), and then, the bindings of φNH-12-5-2 to the immobilized SWNHs in the presence of a peptide were assessed by enzyme-linked immunosorbent assays (ELISAs) (Supporting Information).
Results and Discussion We used a M13 phage library (diversity of ∼2.7 × 109, Ph.D. -12, New England Biolabs, Beverly, MA) that displays a linear 12-mer sequence at the N-terminus of coat protein pIII. The phage has been successfully employed for identifying and isolating peptide aptamers to inorganic materials.8,9 After six rounds of M13 phage library-SWNH panning, we observed an increase in the ratio of bound to input phages (Supporting Information), indicating that SWNH-binding phages were concentrated in the mixture. We selected 33 phage clones from panning cycle 5 and 15 phage clones from panning cycle 6 (total of 48 phage clones), and the pIII tail sequences were identified via standard DNA sequencing techniques. Among the 48 clones, a total of 28 clones (i.e., 15 out of (6) Scott, J. K.; Smith, G. P. Science 1990, 249, 386. (7) (a) Tsang, S. C.; Chen, Y. K.; Harris, P. J. F.; Green, M. L. H. Nature 1994, 372, 159. (b) Kuznetsova, A.; Mawhinney, D. B.; Naumenko, V.; Yates, J. T.; Liu, J.; Smalley, R. E. Chem. Phys. Lett. 2000, 321, 292. (8) (a) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665. (b) Lee, S. W.; Mao, C.; Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892. (c) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nat. Mater. 2002, 1, 169. (d) Sano, K.; Shiba, K. J. Am. Chem. Soc. 2003, 125, 14234. (9) Wang, S.; Humphreys, E.; Chung, S.-Y.; Delduco, D. F.; Lustig, S. R.; Wang, H.; Parker, K. N.; Rizzo, N. W.; Subramoney, S.; Chiang, Y.-M.; Jagota, A. Nat. Mater. 2003, 2, 196.
10.1021/la048968m CCC: $27.50 © 2004 American Chemical Society Published on Web 08/26/2004
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Figure 1. Amino acid sequences that were displayed on SWNHbinding M13 phage pIII tail regions. NH-12-5-2 is the major clone isolated in the panning experiment to SWNHs. NH-125-12 and NH-12-5-1 represent Trp and His (bold letters)-rich minor clones whose amino acid compositions are similar to those observed in the peptide aptamers directed against single-wall carbon nanotubes (CNTs).9 HG-0029 represents the shuffled sequence of NH-12-5-2. Charged and polar residues are colored in red and green, respectively.
the 33 clones from panning cycle 5 and 13 out of the 15 clones from panning cycle 6) have been shown to display an identical peptide sequence, DYFSSPYYEQLF, that is enriched in polar and aromatic residues (NH-12-5-2, Figure 1). Each of the remaining 20 phages displayed distinct pIII peptide tail sequences. However, the majority of these tail sequences were found to be enriched in Trp and His residues (two examples are shown in Figure 1) and are qualitatively similar to the peptide aptamer sequences isolated from phage pannings to single-wall carbon nanotubes (Figure 1).9 From this point on, our analysis will focus on the major aptamer NH-12-5-2 via the use of the cloned phage itself (φNH-12-5-2) and a synthetic peptide representing the DYFSSPYYEQLF sequence. First, we excluded the possibility that the φNH-12-5-2 clones could recognize the non-SWNH portions of SWNH bead conjugates by demonstrating that the φNH-12-5-2 clones do not bind to various control targets including biotin-bound streptavidin-coated magnetic beads and biotin-bound streptavidin-coated ELISA plates (Supporting Information). We then immobilized as-grown SWNHs directly to a plastic plate with ethanol and observed that the φNH-12-5-2 clones exhibited strong binding to SWNHcoated plastic plates (Supporting Information). These results clearly demonstrate that the φNH-12-5-2 clones are recognizing and binding to SWNHs. However, at this time, we cannot distinguish whether the aptamer recognizes and binds to either the pristine graphene structure or the surface composites composed of carbon atoms and other atom(s) that may have been introduced by HNO37 or ethanol treatment.4 This latter possibility is suggested by our previous observations that H2 treatment of O2treated SWNHs, which reduced the polar functional groups on the surfaces, decreased the interaction between φNH-12-5-2 and the modified SWNHs.10 Second, to examine the critical role of the displayed peptide in phage binding, we assessed the competitive effect of the chemically synthesized peptides on the phage binding. Results indicate (Figure 2) that the binding of φNH-12-5-2 to SWNHs was impaired by increasing amounts of free synthetic peptide but not by the control peptide HG-0029 that represents a shuffled sequence version of NH-12-5-2 (Figure 1), indicating that the sequence of NH-12-5-2, and not its amino acid composition, is responsible for the observed binding to SWNHs. Finally, carbon-nanotube-specific peptides have been found to adopt a variety of conformations, such as an R-helix (rationally designed peptide)11 or unfolded, unstructured states (peptide phage selection),9 and it has (10) Zhu, J.; Kase, D.; Shiba, K.; Kasuya, D.; Yudasaka, M.; Iijima, S. Nano Lett. 2003, 3, 1033. (11) Dieckmann, G. R.; Dalton, A. B.; Johnson, P. A.; Razal, J.; Chen, J.; Giordano, G. M.; Munoz, E.; Musselman, I. H.; Baughman, R. H.; Draper, R. K. J. Am. Chem. Soc. 2003, 125, 1770.
Figure 2. Competition assay for the φNH-12-5-2 binding to SWNHs. SWNHs were biotinylated and immobilized on a streptavidin-coated plastic plate (Supporting Information). Phage bindings in the presence of an indicated amount of peptide were assessed at pH 7.5 by the ELISA method (Supporting Information).
Figure 3. CD spectra of NH-12-5-2 at pH 7.6 and pH 2.6 at 5 and 20 °C. The peptide was dissolved in either 100 µM NaH2PO4, pH 2.6, or 100 µM Na2HPO4, pH 7.6, buffer to a final peptide concentration of 12 µM. CD spectra were obtained using an Aviv 60 CD spectrometer, running 60DS software version 4.1t. Peptide samples were scanned using a 1 nm bandwidth and a scan rate of 1 nm/s, with background buffer subtraction performed. The spectrometer was previously calibrated with d-10-camphorsulfphonic acid. A total of three scans were acquired for each peptide sample.
been shown that solution-state and carbon-nanotube (CNT)-bound polypeptide conformations are very similar.11 Thus, to determine the solution-state conformation of NH12-5-2 and set the stage for future solid-state studies of NH-12-5-2 adsorbed onto SWNHs, we employed circular dichroism (CD) spectrometry to study the synthetic DYFSSPYYEQLF peptide under neutral pH and, for comparison, under low pH conditions which would neutralize the C-terminus and D1 and E9 carboxylate side chains (Figure 3). We identified a predominantly randomcoil conformation for this sequence at neutral pH, as evidenced by the presence of π-π* (-) CD absorption bands centered at 198 and 200 nm at 5 and 20 °C, respectively (Figure 3).12 However, an interesting phe(12) (a) Perczel, A.; Hollosi, M.; Sandor, P.; Fasman, G. D. Int. J. Pept. Protein Res. 1993, 41, 223. (b) Sreerama, N.; Woody, R. W. Biochemistry 1994, 33, 10022. (c) Ramirez-Alvarado, M.; Blanco, F. J.; Niemann, H.; Serrano, L. J. Mol. Biol. 1997, 273, 898. (d) Wustman, B. A.; Weaver, J. C.; Morse, D. E.; Evans, J. S. Langmuir 2003, 19, 9373.
Notes
nomenon was observed at pH 2.6; here, the CD spectra for DYFSSPYYEQLF reveal the presence of an R-helical structure existing in equilibrium with a random coil, as evidenced by the presence of (-) absorption bands at 195 nm (π-π*) and 222 nm (n-π*) at 5 °C and a (+) absorption band at 205 nm and a (-) absorption band (n-π*) at 222 nm at 20 °C.11 With the variation in temperature, the changes in position and intensity of the R-helix-specific CD bands at low pH are most likely related to the degree of conformational stability and/or helix distortion in the sequence.13 Obviously, more elaborate techniques involving solution-state and solid-state NMR spectroscopy will be required to confirm whether NH-12-5-2 conformational instability exists in the presence of SWNHs. These studies are currently in progress. Thus, using M13 phage display technology, we have identified a 12-amino-acid (12-AA) pIII tail sequence, DYFSSPYYEQLF, which exhibits a binding preference for SWNH surfaces. This sequence displays an interesting electrostatically driven conformational variation that could well influence its recognition and binding to SWNH surfaces. Additional CD and NMR investigations of these dual conformational states and their potential role in SWNH recognition are currently in progress and will be reported elsewhere. (13) Manning, M. C.; Illangasekare, M.; Woody, R. W. Biophys. Chem. 1988, 31, 77.
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Conclusion Using M13 phage display technology, we have identified a 12-AA pIII tail sequence, DYFSSPYYEQLF, which exhibits a binding preference for SWNH surfaces. This sequence displays an interesting conformational variation that could well influence its recognition and binding to SWNH surfaces: at neutral pH, the sequence adopts a random-coil-like structure, but at low pH where Asp sidechain and R-carboxylate neutralization exists, the sequence prefers an R-helical structure. Additional CD and NMR investigations of these dual conformational states and their potential role in SWNH recognition are currently in progress and will be reported elsewhere. Acknowledgment. We thank Dr. J. Zhu for discussions. J.S.E. acknowledges the support of the Office of Army Research (DAAD19-02-1-0067). Portions of this paper represent contribution 26 from the Lab. for Chem. Phys., N. Y. University. Supporting Information Available: Immobilization of SWNHs, panning experiments, and binding assays. This material is available free of charge via the Internet at http://pubs.acs.org. LA048968M
