The sample surface was immersed in a liquid cell filled with buffer A (,60 mL). In the liquid cell, a small cantilever was fixed. Then, high-speed AFM imaging was performed in buffer A. Here, the diluted samples were used within 3 hours.proteins are at neighboring positions, 400 ml of the solution containing 50 nM CFP-ODN-6 and YFP-ODN-1 in buffer (50 mM Tris-HCl, pH7.5, 10 mM MgCl2, 1 mM dithiothreitol, 100 mM NaCl and 0.01 (w/v) bovine serum albumin) was prepared. For preparation of a protein-DNA complex in which two proteins are at opposite end positions, 400 ml of the solution containing 50 nM CFP-ODN-4 (55 nt), YFP-ODN-1 (55 nt) and other four ODNs (22 (55 nt), 23 (26 nt), 25 (55 nt) and 26 (26 nt)) was prepared. Solution was incubated at 37uC for 1 hour, divided in two halves, and one half was treated with 15 units of EcoRI (Takara, japan) at 37uC for 1 hour. Then fluorescence spectrum was measured (excitation 430 nm/emission 450?600 nm, FP-6500 (Jasco, JAPAN)).ResultsWe used DNA buy GNF-7 structure as a backbone for the designed alignment of several proteins. DNA is widely used to construct nano-architecture by taking advantage of its specific recognition of complementary nucleotide sequences. Recently, enzymes have been aligned on a DNA scaffold to facilitate consecutive reactions [7?4]. The DNA scaffolds in these works are rigid and proteins on the scaffold cannot associate with each other. For our purposes, we designed a DNA backbone composed of five solid segments, each connected by a flexible linker (Fig. 1A). The designed DNAFRET MeasurementCys-CFP conjugated with ODN-4 (55 nt) and ODN-6 (26 nt) and Cys-YFP conjugated with ODN-1 (55 nt) were prepared as above. For preparation of a protein-DNA complex in which twoFlexible Alignment of ProteinFigure 3. Formation of multi-protein-DNA complex. (A) Three representative examples of hybridization of sfGFP-ODNs are shown (B) Various combinations of sfGFP-ODNs, as shown by numbers on top of the lanes, were mixed, hybridized and analyzed by Native-PAGE. GFP fluorescence in the gel was detected. No hybridization occurred for the two leftmost combinations. The rightmost lane shows the products of six sfGFP-ODNs. (C) AFM images of the hybridized product containing six sfGFP-ODNs. Various 58-49-1 arrangements of the six concatenated particles were observed. doi:10.1371/journal.pone.0052534.gFlexible Alignment of ProteinFigure 4. (A) Monomeric (A206K) and (B) dimeric (S208F/V224L) mutant of CFP and YFP were placed at adjacent position (left) and opposite ends (right). Fluorescence spectra were measured without (red) or with (blue) EcoRI treatment. Inset shows intensity ratio of 520 nm/ 480 nm as an indicator of FRET efficiency. The spectra are normalized to the value of 480 nm of the EcoRI-treated sample. doi:10.1371/journal.pone.0052534.gFlexible Alignment of Proteinbackbone consists of six ODNs, of which two have a length of 26 nt and four have a length of 55 nt. For effective hybridization at temperatures safe for proteins, the nucleotide sequences with least propensity for making secondary structure were chosen using the NUPAC web server (http://www.nupack.org/) [15] (table 1). The hybridized structure contains five double-stranded DNA (dsDNA) segments connected by four single-stranded trithymidylate regions. The length of all dsDNA segments is 26 bp, which is approximately equal to 2.5 turns of the helices (8.4 nm), so the 59end of the ODNs in each dsDNA segment is oriented to the same direction. The.The sample surface was immersed in a liquid cell filled with buffer A (,60 mL). In the liquid cell, a small cantilever was fixed. Then, high-speed AFM imaging was performed in buffer A. Here, the diluted samples were used within 3 hours.proteins are at neighboring positions, 400 ml of the solution containing 50 nM CFP-ODN-6 and YFP-ODN-1 in buffer (50 mM Tris-HCl, pH7.5, 10 mM MgCl2, 1 mM dithiothreitol, 100 mM NaCl and 0.01 (w/v) bovine serum albumin) was prepared. For preparation of a protein-DNA complex in which two proteins are at opposite end positions, 400 ml of the solution containing 50 nM CFP-ODN-4 (55 nt), YFP-ODN-1 (55 nt) and other four ODNs (22 (55 nt), 23 (26 nt), 25 (55 nt) and 26 (26 nt)) was prepared. Solution was incubated at 37uC for 1 hour, divided in two halves, and one half was treated with 15 units of EcoRI (Takara, japan) at 37uC for 1 hour. Then fluorescence spectrum was measured (excitation 430 nm/emission 450?600 nm, FP-6500 (Jasco, JAPAN)).ResultsWe used DNA structure as a backbone for the designed alignment of several proteins. DNA is widely used to construct nano-architecture by taking advantage of its specific recognition of complementary nucleotide sequences. Recently, enzymes have been aligned on a DNA scaffold to facilitate consecutive reactions [7?4]. The DNA scaffolds in these works are rigid and proteins on the scaffold cannot associate with each other. For our purposes, we designed a DNA backbone composed of five solid segments, each connected by a flexible linker (Fig. 1A). The designed DNAFRET MeasurementCys-CFP conjugated with ODN-4 (55 nt) and ODN-6 (26 nt) and Cys-YFP conjugated with ODN-1 (55 nt) were prepared as above. For preparation of a protein-DNA complex in which twoFlexible Alignment of ProteinFigure 3. Formation of multi-protein-DNA complex. (A) Three representative examples of hybridization of sfGFP-ODNs are shown (B) Various combinations of sfGFP-ODNs, as shown by numbers on top of the lanes, were mixed, hybridized and analyzed by Native-PAGE. GFP fluorescence in the gel was detected. No hybridization occurred for the two leftmost combinations. The rightmost lane shows the products of six sfGFP-ODNs. (C) AFM images of the hybridized product containing six sfGFP-ODNs. Various arrangements of the six concatenated particles were observed. doi:10.1371/journal.pone.0052534.gFlexible Alignment of ProteinFigure 4. (A) Monomeric (A206K) and (B) dimeric (S208F/V224L) mutant of CFP and YFP were placed at adjacent position (left) and opposite ends (right). Fluorescence spectra were measured without (red) or with (blue) EcoRI treatment. Inset shows intensity ratio of 520 nm/ 480 nm as an indicator of FRET efficiency. The spectra are normalized to the value of 480 nm of the EcoRI-treated sample. doi:10.1371/journal.pone.0052534.gFlexible Alignment of Proteinbackbone consists of six ODNs, of which two have a length of 26 nt and four have a length of 55 nt. For effective hybridization at temperatures safe for proteins, the nucleotide sequences with least propensity for making secondary structure were chosen using the NUPAC web server (http://www.nupack.org/) [15] (table 1). The hybridized structure contains five double-stranded DNA (dsDNA) segments connected by four single-stranded trithymidylate regions. The length of all dsDNA segments is 26 bp, which is approximately equal to 2.5 turns of the helices (8.4 nm), so the 59end of the ODNs in each dsDNA segment is oriented to the same direction. The.
