Mutagenesis and Chemical Reaction. Murali Murugesan ... from time to time. After 1 h, approximately 200 μL of diazonium salt solution was added to 500 μL of.
Supporting Information Virus-based Photo-Responsive Nanowires Formed By Linking Site-Direct Mutagenesis and Chemical Reaction Murali Murugesan, Gopal Abbineni, Susan L. Nimmo, Binrui Cao, and Chuanbin Mao* Department of Chemistry & Biochemistry University of Oklahoma Stephenson Life Sciences Center, 101 Stephenson Parkway Norman, Oklahoma-73019, USA Experimental Section
Amplification & Purification of phage: The engineered phage was amplified in XL1-Blue E.Coli cells. Briefly 5ml of XL1-Blue bacterial culture was incubated with phage in a 1000 ml LB medium and kept in shaking incubator at 37oC for overnight. The cultures were grown at 37 oC in LB medium containing 20g/ml tetracycline, 35g/ml chloramphenicol and 70g/ml of kanamycin and 0.1mM isopropyl--D-thiogalactosidase. The virus particles were purified by double PEG/NaCl precipitation and characterized by TEM. The correct incorporation of the oligonucleotide sequence into the phagemid was confirmed by DNA sequencing result (Fig. S1).
Conjugation of aromatic amine with phage displaying tyrosine-terminated peptide on the side wall to form azo-M13 phage conjugate: Azo-M13 phage conjugate with different aromatic amine end groups (R = -OCH3, -NO2) was made by diazotizing corresponding excess moles of aromatic amine by required sodium nitrite a 0 oC. Further coupling into the tyrosine units on the side wall of the engineered M13 phage via covalent bond formation at 0 oC (Figs. 1 and S2) was done by following a method reported by Schlick etal.1,2 Diazonium salt of corresponding aniline ( 200 μL of 1.5 M solution in acetonitrile) was made by using hydrochloric acid (HCl) (0.81 M; 200 μL ) and 200 μL of 0.4 M aqueous sodium nitrite (NaNO2) in 500 μL of H2O. The resulting mixture was cooled to 0 oC and vortexed from time to time. After 1 h, approximately 200 μL of diazonium salt solution was added to 500 μL of genetically engineered M13 phage solution in 10 mM phosphate buffer with additional 500 μL of borate buffer with 100 mM aqueous sodium chloride solution. Immediate color changes (colorless to yellow, Fig. 3) were
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observed due to the conjugation of diazonium salt in to the engineered M13 phage. Further the reaction mixture was maintained at ~ 5 oC for about 3 h with occasional vortexing. Finally small molecules were removed by dialysis of the azo-M13 phage solution against aqueous phosphate buffer for 36 h using a 50 kDa molecular weight cutoff cellulose dialysis membrane ( Spectrum; Spectrum Labs, USA) with a buffer change performed every 12 h or passed through Sephadex G-25 (Sigma) size exclusion column by using an aqueous phosphate buffer.
Figure S1. DNA sequencing result of the engineered M13 phage, which confirms the display of Y-Y-G-YY-G-Y-Y-G-Y) on the major coat of phage. The red frame highlights the nucleotides corresponding to the displayed sequence.
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Figure S2. (a) Over all reaction scheme indicates the synthesis of methoxy (R=-OCH3) and nitro ended (R=NO2) azo-M13 phage via diazotization reaction between the tyrosine on the side wall of the engineered M13 phage and the aromatic aniline, which results in the formation of new –N=N- link between the p-substituted aromatic amine and ortho position of the tyrosine ring. For the sake of clarity, only a few tyrosines are shown on the side wall of the engineered phage. (b) UV-Vis spectra of engineered M13 phage before (bottom curve) and after (top curves) azo conjugation in water. The broad spectrum of the azo-M13 phage
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in the range of 300-340 nm (for phage with R=-OCH3) and 330-440 nm (for phage with R=-NO2) resulted from the formation of azo chromophore on phage surface.
Figure S3. Change in the absorption spectra (270-480 nm) of nitro-azo-M13 phage (in water) upon exposure to UV light of 365 nm at 5 min different time interval for different times (0-40 min) (top to bottom curves) suggesting the trans-to-cis configurational change of the molecules on the side wall of the phage during light exposure.
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Figure S4. FTIR spectra of the engineered M13 phage (a) and nitro-azo-M13 phage (b) (in solid KBr). Peaks due to the azo linkage –N=N- for the nitro-azo-M13 phage is observed between 1519-1479 cm-1. In case of OCH3 azo M13 phage, –N=N- peaks is observed at 1508 cm-1. The nitro end in NO2-azo-M13 phage identified at ~1519.15 cm-1 and 1343 cm-1 indicates the successful formation of azo chromophore on the phage surface.
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Figure S5. Proton NMR spectra of the engineered M13 phage (a and b) and methoxy-azo-M13 phage (c –d; aromatic region expanded). The spectra indicate the formation of azo chromophore on the ortho position of tyrosine ring of the engineered M 13 phage, which is further confirmed by COSY spectra given in Figure 5.
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References 1 2 3 4
Schlick, T. L., Ding, Z., Kovacs, E. W. & Francis, M. B. Dual surface modification of the tobacco mosaic virus. J. Amer. Chem. Soc. 127, 3718-3723 (2005). Murali, M., Sudhakar, P., Satish, Kumar, B.; Samui, A. B. J Appl Polym Sci , 111, 2562-2573 (2009).,. Liu, W. et al. Enzymatic synthesis of photoactive poly(4-phenylazophenol). Chem. Mater. 12, 1577-1584 (2000). Buruian, E. C., Buruian, T., Airinei, A. & Robil, G. Photochromism of polyurethane cationomers with pendent azo groups Angewandte Makromolekulare Chemie 206, 87-96 (1993).
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