- Email: [email protected]
In vitro selection of peptide aptamers using a ribosome display for a conducting polymer
Journal of Bioscience and Bioengineering VOL. 117 No. 4, 501e503, 2014 www.elsevier.com/locate/jbiosc
NOTE
In vitro selection of peptide aptamers using a ribosome display for a conducting polymer Zha Li,1, 2, 3 Takanori Uzawa,1, 2 Haichao Zhao,4 Shyh-Chyang Luo,4 Hsiao-hua Yu,4 Eiry Kobatake,3 and Yoshihiro Ito1, 2, 3, * Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan,1 Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan,2 Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 1259 Nagatsuda, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan,3 and Responsive Organic Materials Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan4 Received 1 July 2013; accepted 4 October 2013 Available online 5 November 2013
Ribosome display was used to select peptide aptamers from a random library composed of hydrophilic amino acids for a conducting polymer, poly(3-hexylthiophene-2,5-diyl). Binding of aptamers was measured by quartz crystal microbalance, and the secondary structure of the peptide was investigated by circular dichroism. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: In vitro selection; Ribosome display; Poly(3-hexylthiophene-2,5-diyl); Peptide aptamer; Binding ability]
To create new functional materials, combining of different components is important; therefore, researchers have designed molecules that bind inorganic and organic materials. The screening of such molecules using in vitro selection has been conducted, with peptide aptamers reported for different kinds of non-biological materials including metal oxides (1), metals (2), quantum dots (3), and synthetic polymers (4e6). These peptides are usually selected by phage display. However, these displays have drawbacks, such as limited diversity and steric hindrance of phages. Although T7 phage display system was developed to overcome the problems, nonbiological display system provides more diverse and no limitation due to toxicity. In our work, we used a ribosome display for the in vitro selection of peptide aptamers for the polymer, poly(3-hexylthiophene-2,5diyl) (P3HT). P3HT belongs to a family of important semiconducting polymers that are used in diverse applications, such as solar cells (7), transistors, and sensors (8). The peptide aptamer will be useful for preparation of biosensors to combine the P3HT and biological molecules. Although ribosome displays have not been used for such non-biological targets, we previously found that this method could be applied to carbon nanotubes (9). Therefore, we used a ribosome display for selecting peptide aptamers that can bind to P3HT. We designed a single-stranded DNA library (Operon, Tokyo, Japan) that contained (VVN)10 random sequences to prepare a peptide library composed of hydrophilic acids only (supplementary * Corresponding author at: Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel.: þ81 48 467 5809; fax: þ81 48 467 9300. E-mail addresses: [email protected], [email protected] (Y. Ito).
data, Table S1). It is believed that pep stacking interaction is important between the peptides and the polymer. Therefore, we attempted to seek the possibility of peptide and polymer interaction without the pep interaction. At the same time, we expected to select aqueous soluble peptides by removal of hydrophobic amino acids. The selection method was performed as previously reported (supplementary data, Fig. S1) (9). Initially, one round of polymerase chain reaction (PCR) amplification was conducted using a GXL enzyme kit (Qiagen, Tokyo, Japan). This resulted in double-stranded DNA sequences, which were subsequently digested by SfiI (New England Biolabs, Ipswich, MA, USA) and ligated into the 13Trx plasmid vector (Fig. S2). Fifteen pmol of random sequence DNA (1013 diversity) was ligated to plasmid and amplified by 10 rounds of PCR. Using the PURE system (Wako Pure Chemicals, Osaka, Japan), in vitro transcription and translation was conducted to form the peptideeribosomeemRNA complex. P3HT was synthesized by Grignard metathesis polymerization as previously reported (10). The molecular weight and PDI were about 14,000 and 1.6, respectively. The prepared P3HT powder was immersed in ethanol and the suspension centrifuged at 20,000 g with laptop centrifuge machine Himac CT15 (Hitachi Koki Co. Ltd, Minato-ku, Tokyo, Japan). The precipitated P3HT powder was washed 6 times with water and mixed with the 50 mL solution from PURE system containing 60 pmol peptideeribosomeemRNA complex. After incubation in 600 mL buffer (0.1% Tween 20, 60 mM Tris, 180 mM NaCl, 60 mM magnesium acetate) at 4 C for 1 h, the suspension was centrifuged and washed with water six times. The 100 mL solution containing sediment of P3HT/peptideeribosomeemRNA complex 0.1 w/v % was eluted with 10 mL of 10 mM EDTA to release mRNA. The mixture was centrifuged at
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.10.005
502
LI ET AL.
J. BIOSCI. BIOENG.,
TABLE 1. Sequences of selected peptides. Name of peptide S1 S3 S5 S6
Sequence DGDRNQSSGG GGTQSDGTRG DPRGRGESGR HGTSKGHTRD
Copies 2 2 2 2
FIG. 1. The final binding mass of different peptides [S1, S3, S6, TSA (MQASNEGQL), RGD (GRGDSP) and P1 (HWSAWWIRSNOS)] at 1 mM for P3HT film in a pH 7.4 buffer by QCM.
20,000 g and the supernatant containing mRNA was drawn out and purified by RNeasy kits. The mRNA was collected for in vitro reverse transcription and the resulting cDNA was amplified. These processes were repeated as one round. After 7 and 9 rounds, 96 clones were sequenced from the resulting cDNAs. However, no overlapping sequences were found. After 11 rounds, four duplicated sequences were found in the 96 clones. Therefore, the process of selection was finalized. The four sequences (Table 1) were synthesized by ProImmune Co. Ltd. (Sarasota, FL, USA). These four peptides comprised hydrophilic amino acids, but the S5 peptide was found to be insoluble in water. A possible reason for this could be strong intra-molecular interactions between S5 peptides resulting in aggregation and insolubility in water (11). The S5 peptide contained five amino acids with cationic/anionic side chains (1E, 1D and 3R) suggesting that strong intra-molecular interactions were likely to occur.
We measured the binding ability of the three soluble peptides. The binding ability of the peptides was analyzed with a quartz crystal microbalance (QCM; Q-sense D300, Gothenburg, Sweden). P3HT was dissolved in THF (1 g/L) and was spin-coated on the QCM crystal chips by 5000 rpm for 50 s. And the peptide solutions in Tris-buffered saline (1 mg/mL) were flowed over the P3HT-coated chips. Changes in the QCM frequency indicated the amount of peptide bound to the polymer, and the frequency change data was converted into binding mass using the Qtools software package. The binding mass increased for around 1000 s. After 1000 s, the buffer solution began entering the chip-installed cell and washed the peptide away. After the washing, although binding mass of the selected peptides (S1, S3, and S6) was observed, no binding of RGD (GRGDSP) or TSA peptide (MQASNEGQL) which is composed of only hydrophilic amino acids was found (Fig. 1). P1 peptide (HWSAWWIRSNOS, 12) which was reported to bind carbon nanotube and contains aromatic amino acids bound to P3HT and the level was comparable to S1 (Fig. 1). It has been reported that in the interactions between peptides and polymers (4,5), pep stacking between the aromatic rings of the peptides and polymers plays an important role as also found in this study of P1 binding on P3HT. However, typical amino acids with aromatic components that may contribute to pep stacking, such as phenylalanine, tyrosine, and tryptophan, were excluded from our library. Histidine was only present in S6, and showed similar binding ability to S1 and less than S3. Our findings indicate that pep stacking interactions were not essential for the binding of these peptides to P3HT, although pep stacking between P3HT and S6 cannot be excluded as this peptide contains a histidine residue. As previously reported (13), it is possible that target-inducing conformational change of peptides could optimize hydrophobic interactions between the peptide and P3HT to achieve greater binding affinity. Circular dichroism (CD) spectra were measured with a JASCO J720 (JASCO, Hachioji, Tokyo, Japan). We used a quartz cell with a 1 mm path length, a scan speed of 200 nm/min, 16 accumulations, a 2.0 nm bandwidth, and a response time of 1 s. The peptide concentration measured was 100 mM. Fig. 2 shows that all the selected peptides had no specific secondary structures, while RGD and P1 contained b turns (14) and a-helix (12), respectively, as reported previously. Considering that the selected peptides had no specific secondary structure, the binding affinity of peptides may be attributed to induced fitting. The ribosome display can be successfully used for selection of hydrophilic peptide aptamers against P3HT. The peptides show specific binding abilities, as determined by QCM. While it is commonly believed that pep stacking and hydrophobic interactions are necessary for the peptides and hydrophobic polymer, we have shown that the peptides consisting of only hydrophilic amino acids have good binding ability to a hydrophobic polymer.
FIG. 2. CD spectra of the S1, S3, S6, TSA, RGD and P1 peptides.
VOL. 117, 2014 Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.10.005. This work was supported by JSPS KAKENHI (Grant Number 22220009).
References 1. Brown, S.: Engineered iron oxide-adhesion mutants of the Escherichia coli phage l receptor, Proc. Natl. Acad. Sci. USA, 89, 8651e8655 (1992). 2. Brown, S.: Metal-recognition by repeating polypeptides, Nat. Biotechnol., 15, 269e272 (1997). 3. Whaley, S. R., English, D. S., Hu, E. L., Barbara, P. F., and Belcher, A. M.: Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly, Nature, 405, 665e668 (2000). 4. Ejima, H., Matsumiya, K., Sawada, T., and Serizawa, T.: Conjugated polymer nanoparticles hybridized with the peptide aptamer, Chem. Commun., 47, 7707e7709 (2011). 5. Ejima, H., Matsuno, H., and Serizawa, T.: Biological identification of peptides that specifically bind to poly(phenylene vinylene) surfaces: recognition of the branched or linear structure of the conjugated polymer, Langmuir, 26, 17278e17285 (2010). 6. Sanghvi, A. B., Miller, K. P. H., Belcher, A. M., and Schmidt, C. E.: Biomaterials functionalization using a novel peptide that selectively binds to a conducting polymer, Nat. Mater., 4, 496e502 (2005).
NOTE
503
7. Brabec, C. J., Gowrisanker, S., Halls, J. J. M., Laird, D., Jia, S. J., and Williams, S. P.: Polymerefullerene bulk-heterojunction solar cells, Adv. Mater., 22, 3839e3856 (2010). 8. Wei, F. and Ho, C. M.: Aptamer-based electrochemical biosensor for botulinum neurotoxin, Anal. Bioanal. Chem., 393, 1943e1948 (2009). 9. Li, Z., Uzawa, T., Tanaka, T., Hida, A., Ishibashi, K., Katakura, H., Kobatake, E., and Ito, Y.: In vitro selection of peptide aptamers with affinity to single-wall carbon nanotubes using a ribosome display, Biotechnol. Lett., 35, 39e45 (2013). 10. Loewe, R. S., Khersonsky, S. M., and McCullough, R. D.: A simple method to prepare head-to-tail coupled, regioregular poly(3-alkylthiophenes) using Grignard metathesis, Adv. Mater., 11, 250e253 (1999). 11. Mayo, K. H., Ilyina, E., and Park, H.: A recipe for designing water-soluble, betasheet-forming peptides, Protein Sci., 5, 1301e1315 (1996). 12. Wang, S., Humphrevs, E., Chung, S., Delduco, D., Lustig, S., Wang, H., Parker, K., Rizzo, N., Subramoney, S., Chiang, Y., and Jagota, A.: Peptides with selective affinity for carbon nanotubes, Nat. Mater., 2, 196e200 (2003). 13. Mermut, O., Phillips, D. C., York, R. L., McCrea, K. R., Ward, R. S., and Somorjai, G. A.: In situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic polystyrene and hydrophilic silica surfaces using quartz crystal microbalance, atomic force microscopy, and sum frequency generation vibrational spectroscopy, J. Am. Chem. Soc., 128, 3598e3607 (2006). 14. Reed, J., Hull, W. E., Vonderlieth, C. W., Kubler, D., Suhai, S., and Kinzel, V.: Secondary structure of the Arg-Gly-Asp recognition site in proteins involved in cell-surface adhesion e evidence for the occurrence of nested beta-bends in the model hexapeptide GRGDSP, Eur. J. Biochem., 178, 141e154 (1988).
NOTE
In vitro selection of peptide aptamers using a ribosome display for a conducting polymer Zha Li,1, 2, 3 Takanori Uzawa,1, 2 Haichao Zhao,4 Shyh-Chyang Luo,4 Hsiao-hua Yu,4 Eiry Kobatake,3 and Yoshihiro Ito1, 2, 3, * Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan,1 Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan,2 Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 1259 Nagatsuda, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan,3 and Responsive Organic Materials Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan4 Received 1 July 2013; accepted 4 October 2013 Available online 5 November 2013
Ribosome display was used to select peptide aptamers from a random library composed of hydrophilic amino acids for a conducting polymer, poly(3-hexylthiophene-2,5-diyl). Binding of aptamers was measured by quartz crystal microbalance, and the secondary structure of the peptide was investigated by circular dichroism. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: In vitro selection; Ribosome display; Poly(3-hexylthiophene-2,5-diyl); Peptide aptamer; Binding ability]
To create new functional materials, combining of different components is important; therefore, researchers have designed molecules that bind inorganic and organic materials. The screening of such molecules using in vitro selection has been conducted, with peptide aptamers reported for different kinds of non-biological materials including metal oxides (1), metals (2), quantum dots (3), and synthetic polymers (4e6). These peptides are usually selected by phage display. However, these displays have drawbacks, such as limited diversity and steric hindrance of phages. Although T7 phage display system was developed to overcome the problems, nonbiological display system provides more diverse and no limitation due to toxicity. In our work, we used a ribosome display for the in vitro selection of peptide aptamers for the polymer, poly(3-hexylthiophene-2,5diyl) (P3HT). P3HT belongs to a family of important semiconducting polymers that are used in diverse applications, such as solar cells (7), transistors, and sensors (8). The peptide aptamer will be useful for preparation of biosensors to combine the P3HT and biological molecules. Although ribosome displays have not been used for such non-biological targets, we previously found that this method could be applied to carbon nanotubes (9). Therefore, we used a ribosome display for selecting peptide aptamers that can bind to P3HT. We designed a single-stranded DNA library (Operon, Tokyo, Japan) that contained (VVN)10 random sequences to prepare a peptide library composed of hydrophilic acids only (supplementary * Corresponding author at: Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel.: þ81 48 467 5809; fax: þ81 48 467 9300. E-mail addresses: [email protected], [email protected] (Y. Ito).
data, Table S1). It is believed that pep stacking interaction is important between the peptides and the polymer. Therefore, we attempted to seek the possibility of peptide and polymer interaction without the pep interaction. At the same time, we expected to select aqueous soluble peptides by removal of hydrophobic amino acids. The selection method was performed as previously reported (supplementary data, Fig. S1) (9). Initially, one round of polymerase chain reaction (PCR) amplification was conducted using a GXL enzyme kit (Qiagen, Tokyo, Japan). This resulted in double-stranded DNA sequences, which were subsequently digested by SfiI (New England Biolabs, Ipswich, MA, USA) and ligated into the 13Trx plasmid vector (Fig. S2). Fifteen pmol of random sequence DNA (1013 diversity) was ligated to plasmid and amplified by 10 rounds of PCR. Using the PURE system (Wako Pure Chemicals, Osaka, Japan), in vitro transcription and translation was conducted to form the peptideeribosomeemRNA complex. P3HT was synthesized by Grignard metathesis polymerization as previously reported (10). The molecular weight and PDI were about 14,000 and 1.6, respectively. The prepared P3HT powder was immersed in ethanol and the suspension centrifuged at 20,000 g with laptop centrifuge machine Himac CT15 (Hitachi Koki Co. Ltd, Minato-ku, Tokyo, Japan). The precipitated P3HT powder was washed 6 times with water and mixed with the 50 mL solution from PURE system containing 60 pmol peptideeribosomeemRNA complex. After incubation in 600 mL buffer (0.1% Tween 20, 60 mM Tris, 180 mM NaCl, 60 mM magnesium acetate) at 4 C for 1 h, the suspension was centrifuged and washed with water six times. The 100 mL solution containing sediment of P3HT/peptideeribosomeemRNA complex 0.1 w/v % was eluted with 10 mL of 10 mM EDTA to release mRNA. The mixture was centrifuged at
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.10.005
502
LI ET AL.
J. BIOSCI. BIOENG.,
TABLE 1. Sequences of selected peptides. Name of peptide S1 S3 S5 S6
Sequence DGDRNQSSGG GGTQSDGTRG DPRGRGESGR HGTSKGHTRD
Copies 2 2 2 2
FIG. 1. The final binding mass of different peptides [S1, S3, S6, TSA (MQASNEGQL), RGD (GRGDSP) and P1 (HWSAWWIRSNOS)] at 1 mM for P3HT film in a pH 7.4 buffer by QCM.
20,000 g and the supernatant containing mRNA was drawn out and purified by RNeasy kits. The mRNA was collected for in vitro reverse transcription and the resulting cDNA was amplified. These processes were repeated as one round. After 7 and 9 rounds, 96 clones were sequenced from the resulting cDNAs. However, no overlapping sequences were found. After 11 rounds, four duplicated sequences were found in the 96 clones. Therefore, the process of selection was finalized. The four sequences (Table 1) were synthesized by ProImmune Co. Ltd. (Sarasota, FL, USA). These four peptides comprised hydrophilic amino acids, but the S5 peptide was found to be insoluble in water. A possible reason for this could be strong intra-molecular interactions between S5 peptides resulting in aggregation and insolubility in water (11). The S5 peptide contained five amino acids with cationic/anionic side chains (1E, 1D and 3R) suggesting that strong intra-molecular interactions were likely to occur.
We measured the binding ability of the three soluble peptides. The binding ability of the peptides was analyzed with a quartz crystal microbalance (QCM; Q-sense D300, Gothenburg, Sweden). P3HT was dissolved in THF (1 g/L) and was spin-coated on the QCM crystal chips by 5000 rpm for 50 s. And the peptide solutions in Tris-buffered saline (1 mg/mL) were flowed over the P3HT-coated chips. Changes in the QCM frequency indicated the amount of peptide bound to the polymer, and the frequency change data was converted into binding mass using the Qtools software package. The binding mass increased for around 1000 s. After 1000 s, the buffer solution began entering the chip-installed cell and washed the peptide away. After the washing, although binding mass of the selected peptides (S1, S3, and S6) was observed, no binding of RGD (GRGDSP) or TSA peptide (MQASNEGQL) which is composed of only hydrophilic amino acids was found (Fig. 1). P1 peptide (HWSAWWIRSNOS, 12) which was reported to bind carbon nanotube and contains aromatic amino acids bound to P3HT and the level was comparable to S1 (Fig. 1). It has been reported that in the interactions between peptides and polymers (4,5), pep stacking between the aromatic rings of the peptides and polymers plays an important role as also found in this study of P1 binding on P3HT. However, typical amino acids with aromatic components that may contribute to pep stacking, such as phenylalanine, tyrosine, and tryptophan, were excluded from our library. Histidine was only present in S6, and showed similar binding ability to S1 and less than S3. Our findings indicate that pep stacking interactions were not essential for the binding of these peptides to P3HT, although pep stacking between P3HT and S6 cannot be excluded as this peptide contains a histidine residue. As previously reported (13), it is possible that target-inducing conformational change of peptides could optimize hydrophobic interactions between the peptide and P3HT to achieve greater binding affinity. Circular dichroism (CD) spectra were measured with a JASCO J720 (JASCO, Hachioji, Tokyo, Japan). We used a quartz cell with a 1 mm path length, a scan speed of 200 nm/min, 16 accumulations, a 2.0 nm bandwidth, and a response time of 1 s. The peptide concentration measured was 100 mM. Fig. 2 shows that all the selected peptides had no specific secondary structures, while RGD and P1 contained b turns (14) and a-helix (12), respectively, as reported previously. Considering that the selected peptides had no specific secondary structure, the binding affinity of peptides may be attributed to induced fitting. The ribosome display can be successfully used for selection of hydrophilic peptide aptamers against P3HT. The peptides show specific binding abilities, as determined by QCM. While it is commonly believed that pep stacking and hydrophobic interactions are necessary for the peptides and hydrophobic polymer, we have shown that the peptides consisting of only hydrophilic amino acids have good binding ability to a hydrophobic polymer.
FIG. 2. CD spectra of the S1, S3, S6, TSA, RGD and P1 peptides.
VOL. 117, 2014 Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2013.10.005. This work was supported by JSPS KAKENHI (Grant Number 22220009).
References 1. Brown, S.: Engineered iron oxide-adhesion mutants of the Escherichia coli phage l receptor, Proc. Natl. Acad. Sci. USA, 89, 8651e8655 (1992). 2. Brown, S.: Metal-recognition by repeating polypeptides, Nat. Biotechnol., 15, 269e272 (1997). 3. Whaley, S. R., English, D. S., Hu, E. L., Barbara, P. F., and Belcher, A. M.: Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly, Nature, 405, 665e668 (2000). 4. Ejima, H., Matsumiya, K., Sawada, T., and Serizawa, T.: Conjugated polymer nanoparticles hybridized with the peptide aptamer, Chem. Commun., 47, 7707e7709 (2011). 5. Ejima, H., Matsuno, H., and Serizawa, T.: Biological identification of peptides that specifically bind to poly(phenylene vinylene) surfaces: recognition of the branched or linear structure of the conjugated polymer, Langmuir, 26, 17278e17285 (2010). 6. Sanghvi, A. B., Miller, K. P. H., Belcher, A. M., and Schmidt, C. E.: Biomaterials functionalization using a novel peptide that selectively binds to a conducting polymer, Nat. Mater., 4, 496e502 (2005).
NOTE
503
7. Brabec, C. J., Gowrisanker, S., Halls, J. J. M., Laird, D., Jia, S. J., and Williams, S. P.: Polymerefullerene bulk-heterojunction solar cells, Adv. Mater., 22, 3839e3856 (2010). 8. Wei, F. and Ho, C. M.: Aptamer-based electrochemical biosensor for botulinum neurotoxin, Anal. Bioanal. Chem., 393, 1943e1948 (2009). 9. Li, Z., Uzawa, T., Tanaka, T., Hida, A., Ishibashi, K., Katakura, H., Kobatake, E., and Ito, Y.: In vitro selection of peptide aptamers with affinity to single-wall carbon nanotubes using a ribosome display, Biotechnol. Lett., 35, 39e45 (2013). 10. Loewe, R. S., Khersonsky, S. M., and McCullough, R. D.: A simple method to prepare head-to-tail coupled, regioregular poly(3-alkylthiophenes) using Grignard metathesis, Adv. Mater., 11, 250e253 (1999). 11. Mayo, K. H., Ilyina, E., and Park, H.: A recipe for designing water-soluble, betasheet-forming peptides, Protein Sci., 5, 1301e1315 (1996). 12. Wang, S., Humphrevs, E., Chung, S., Delduco, D., Lustig, S., Wang, H., Parker, K., Rizzo, N., Subramoney, S., Chiang, Y., and Jagota, A.: Peptides with selective affinity for carbon nanotubes, Nat. Mater., 2, 196e200 (2003). 13. Mermut, O., Phillips, D. C., York, R. L., McCrea, K. R., Ward, R. S., and Somorjai, G. A.: In situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic polystyrene and hydrophilic silica surfaces using quartz crystal microbalance, atomic force microscopy, and sum frequency generation vibrational spectroscopy, J. Am. Chem. Soc., 128, 3598e3607 (2006). 14. Reed, J., Hull, W. E., Vonderlieth, C. W., Kubler, D., Suhai, S., and Kinzel, V.: Secondary structure of the Arg-Gly-Asp recognition site in proteins involved in cell-surface adhesion e evidence for the occurrence of nested beta-bends in the model hexapeptide GRGDSP, Eur. J. Biochem., 178, 141e154 (1988).