Passage
The HIV-derived transactivator of transcription (Tat) is a short peptide with the sequence RKKRRQRRRPPQ. When paired with liposomes (ie, synthetic vesicles) composed of cationic lipids, Tat can transport large molecules across cell membranes and has been studied as a vector for gene therapy. However, Tat delivers cargo to cells with relatively low efficiency. Researchers hypothesized that a Tat homodimer could yield stronger DNA binding than the monomer, which may improve the efficiency of DNA delivery.To study this hypothesis, Tat and two variant peptides (Variants 1 and 2) were produced by solid-phase peptide synthesis, in which the growing peptide chain is immobilized on polystyrene beads. Unlike biological systems, solid-phase synthesis assembles peptides from the C-terminus to the N-terminus, as shown in Figure 1.
Figure 1 Simplified schematic of one round of solid-phase peptide synthesis. The protecting group is removed during deprotection. The process is repeated until the peptide is complete.Variants 1 and 2 contained a C-terminal and an N-terminal cysteine residue, respectively. Each variant was allowed to dimerize by disulfide bond formation. Peptide-DNA binding interactions were then studied by mixing varying amounts of each peptide with plasmid DNA encoding luciferase protein (pLuc) . The resulting peptide-DNA complexes were run on agarose gels, with results displayed in Figure 2.
Figure 2 Agarose gel results for pLuc plasmid exposed to varying amounts of each peptideEqual concentrations of each type of peptide-DNA complex were then incubated with cationic liposomes and exposed to cultured cells for 24 hours. The cells were then assessed for luciferase activity. Greater luciferase activity indicated more efficient delivery of the pLuc plasmid. The results are shown in Figure 3.
Figure 3 Plasmid delivery efficiency of three peptides, as measured by induced luciferase activity
Adapted from Lee S-J, Yoon S-H, Doh K-O. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond. J Microbiol Biotechnol. 2011;21(8) :802-807.
-Do the data in Figure 2 and Figure 3 support the hypothesis that stronger peptide-DNA interactions yield more efficient plasmid delivery?

Question

Passage
The HIV-derived transactivator of transcription (Tat) is a short peptide with the sequence RKKRRQRRRPPQ. When paired with liposomes (ie, synthetic vesicles) composed of cationic lipids, Tat can transport large molecules across cell membranes and has been studied as a vector for gene therapy. However, Tat delivers cargo to cells with relatively low efficiency. Researchers hypothesized that a Tat homodimer could yield stronger DNA binding than the monomer, which may improve the efficiency of DNA delivery.To study this hypothesis, Tat and two variant peptides (Variants 1 and 2) were produced by solid-phase peptide synthesis, in which the growing peptide chain is immobilized on polystyrene beads. Unlike biological systems, solid-phase synthesis assembles peptides from the C-terminus to the N-terminus, as shown in Figure 1.

Figure 1 Simplified schematic of one round of solid-phase peptide synthesis. The protecting group is removed during deprotection. The process is repeated until the peptide is complete.Variants 1 and 2 contained a C-terminal and an N-terminal cysteine residue, respectively. Each variant was allowed to dimerize by disulfide bond formation. Peptide-DNA binding interactions were then studied by mixing varying amounts of each peptide with plasmid DNA encoding luciferase protein (pLuc) . The resulting peptide-DNA complexes were run on agarose gels, with results displayed in Figure 2.

Figure 2 Agarose gel results for pLuc plasmid exposed to varying amounts of each peptideEqual concentrations of each type of peptide-DNA complex were then incubated with cationic liposomes and exposed to cultured cells for 24 hours. The cells were then assessed for luciferase activity. Greater luciferase activity indicated more efficient delivery of the pLuc plasmid. The results are shown in Figure 3.

Figure 3 Plasmid delivery efficiency of three peptides, as measured by induced luciferase activity
Adapted from Lee S-J, Yoon S-H, Doh K-O. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond. J Microbiol Biotechnol. 2011;21(8) :802-807.
-Do the data in Figure 2 and Figure 3 support the hypothesis that stronger peptide-DNA interactions yield more efficient plasmid delivery?

Quizplus · Accepted Answer

11ecba2d_0f29_6b7d_a3bf_e3372b1b73c3_MD0008_00 DNA molecules have a net negative charge due to the phosphate groups in the DNA backbone. This negative charge causes DNA to migrate toward the anode of an electrophoretic cell through an agarose gel. Neutralizing some of the negative charges by binding to positively charged amino acid residues decreases the mobility of the DNA through the gel. Peptide binding also increases the mass of the complex, further slowing its migration. As more charges are neutralized and more mass is added, the DNA migrates progressively shorter distances through the gel. Figure 2 shows that all three forms of the Tat peptide (which are positively charged) cause a decrease in gel mobility. However, different amounts of each form are required to achieve an effect, and the relative amounts needed indicate binding strength. The Variant 1 dimer shows a significant decrease in DNA mobility when 0.5 nM peptide is added. The Variant 2 dimer shows a small mobility shift at 0.5 nM but requires 1 nM peptide for a significant effect. The Tat monomer shows no effect at 0.5 nM. Therefore, Variant 1 shows the strongest binding, followed by Variant 2, and then the Tat monomer. Figure 3 shows that Variant 1 yields the most efficient DNA delivery, followed by Variant 2, and then the Tat monomer. Therefore, the order of binding strength is the same as the order of delivery efficiency. This supports the hypothesis that stronger peptide-DNA binding is associated with increased delivery efficiency by Tat peptides. (Choice B) Variant 2 shows stronger (not weaker) binding than the Tat monomer. (Choice C) The Tat monomer shows the weakest (not strongest) binding. (Choice D) Variant 2 binds DNA with less (not greater) strength than Variant 1. Educational objective: The negative charge of DNA causes it to migrate toward the anode of an electrophoretic cell. Neutralizing the negative charge with positively charged residues and increasing the size of the complex decreases the DNA's gel mobility.

Passage The HIV-Derived Transactivator of Transcription (Tat) Is a Short Peptide