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1.
Figure 9.

Figure 9. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Transmission electron micrographs of phi29 procapsids/pRNA-gold complexes. Bar = 50 nm.

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.
2.
Figure 7.

Figure 7. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Sucrose gradient sedimentation to compare the binding efficiency of pRNA and tRNA to procapsid. The [3H] pRNA Cd′ (black square), [3H] Cd′ + Dc′ (open triangle) and tRNA (asterisk) (2.5 pmol each) were incubated with procapsid and then subjected to 5–20% sucrose gradient sedimentation.

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.
3.
Figure 5.

Figure 5. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Histogram showing the photobleaching steps of the procapsids containing fluorescence pRNA in single molecule counting using the single molecule total internal reflection florescence dual view system (44,72) (Animations available at: http://www.eng.uc.edu/nanomedicine/newmovs.html).

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.
4.
Figure 6.

Figure 6. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Comparison of the activity of DNA-packaging (A) and virion assembly (B) of different RNAs. The packaged DNA was protected from DNase I digestion and was indicated in the gel (lanes 6–9 in A). Lane 2 is the input phi29 DNA control without DNase I digestion.

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.
5.
Figure 3.

Figure 3. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Agarose gel shift assay to compare the binding affinity of connector with individual pRNA monomer Cd′ or tRNA. A final concentration of 0.5 µM purified connector was mixed with an increasing concentration of pRNA monomer Cd′ or nonspecific tRNA. The triangle indicates a 2-fold increase in RNA concentration. The complex was separated by 0.8% agarose gel. The gel was first stained with ethidium bromide to detect RNA (A) and then stained by Coomassie brilliant blue to detect the protein (B).

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.
6.
Figure 1.

Figure 1. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Secondary structure of pRNA and hexamer formation. (A) Side-view of phi29 procapsid. (B) Illustration of right- and left-hand interaction via the interlocking right loop A (5′GGAC) and left loop a′ (3′CCUG) to form a pRNA hexamer. The connector binding domain is shaded. (C) Sequence and secondary structure of pRNA Aa′. The lightly boxed regions indicate the location for the truncation and insertion of nucleotide to construct mutant pRNAs that form into pRNA hexameric rings with reduced or enlarged diameter, respectively.

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.
7.
Figure 2.

Figure 2. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Single molecule imaging for the comparison of pRNA oligomerization in the presence (A) and in the absence (B) of procapsid. (a) Monomeric pRNA Ab′; (b) dimeric pRNA Aa′; and (C) pRNA with the deletion of 2-nt pairs to reduce the diameter of the hexameric ring. Each bright spot represents one single procapsid/Cy3-pRNA complex (A) or the Cy3-pRNA complex in the absence of procapsid or connector (B). The histogram represents the photobleaching steps of the complex that contained the Cy3-labeled RNA. The measurements and pRNA counting have been performed using the single molecule total internal reflection florescence dual view system (44,72).

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.
8.
Figure 8.

Figure 8. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Single molecule imaging to compare the procapsid-binding efficiency between the ring forming pRNA group (A) and the pRNA group with one of the interlocking links disrupted (B). The binding of procapsid with pRNA monomer Cd′ (C), dsDNA (D) and ssDNA (E) were also shown as controls. The pRNA Cd′ was labeled with a single Cy3 molecule. Cy3-ssDNA was synthesized by IDT. The procapsid/Cy3-RNA or procapsid/Cy3-DNA complex, which was purified from 5–20% sucrose gradient and attached to the quartz slide surface coated with anti-phi29 procapsid antibody. (A) Procapsid/Cy3-pRNA(Cd′ + Dc′); (B) procapsid/Cy3-pRNA(Cd′ + De′) with one link disrupted; (C) procapsid/Cy3-pRNA(Cd); (D) procapsid/Cy3-dsDNA; and (E) procapsid/Cy3-ssDNA.

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.
9.
Figure 4.

Figure 4. From: Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging.

Single molecule counting of RNA molecules that bound to each procapsid (44,72). (AE) Single molecule imaging of procapsid/Cy3-RNA complex. Each bright spot represents one single procapsid/Cy3-pRNA complex. Cy3 signals were presented in a pseudo color, green. (A) complexes of procapsid/Cy3-pRNA (Cd′ + Dc′); (B) procapsid/Cy3-pRNA (Cd′); (C) procapsid/Cy3-tRNA; (D) procapsid/Cy3-pRNA (Aa′); and (E) procapsid/Cy3-pRNA (Yy′). (FJ). Typical plot of fluorescence intensity versus time for Cy3-pRNA in procapsid/pRNA complexes. Each step in photobleaching represents the presence of one Cy3-RNA, which was labeled with a single Cy3-fluorophore. Data of photobleaching steps were obtained using methods in our recent publications (44,72). Examples of the histogram distribution are shown in Figure 5. Statistical analysis (44) revealed that the resulting data were highly significant. The ‘||’ at the vertices of the inserted diagram indicates that two loops were noncomplementary and could not interlock to form a ring. Cy3-labeled RNA is shown as a green line, while unlabeled RNA is shown as a black line in the inserted diagram (Animations available at: http://www.eng.uc.edu/nanomedicine/newmovs.html).

Feng Xiao, et al. Nucleic Acids Res. 2008 November;36(20):6620-6632.

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