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.
-A researcher hypothesized that cargo delivery is most efficient when the peptide-DNA complex forms prior to DNA interactions with liposomes. Which additional observation would best support this hypothesis?

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.
-A researcher hypothesized that cargo delivery is most efficient when the peptide-DNA complex forms prior to DNA interactions with liposomes. Which additional observation would best support this hypothesis?

Quizplus · Accepted Answer

11ecba2d_0f2a_a3fe_a3bf_3dca91e93374_MD0008_00 Biological complexes consisting of multiple molecules often must assemble in a particular order. In the case of Tat, DNA, and liposomes, a researcher hypothesized that Tat or its variants must bind to DNA before DNA interacts with cationic liposomes to yield optimal DNA delivery. If this hypothesis is correct, then DNA binding to cationic liposomes prior to interactions with peptides would result in decreased DNA delivery. Figure 3 was generated by mixing existing peptide-DNA complexes (ie, the peptide and DNA are already interacting) with liposomes and then incubating with cells. Mixing DNA with cationic liposomes prior to addition of peptides would allow DNA-liposome interactions to occur before DNA can interact with peptides. If such interactions interfere with DNA delivery (as the researcher suggests), then less luciferase will be produced and less activity will be observed. This observation would provide strong support for the researcher's hypothesis. (Choices A and C) Increasing the concentration of liposomes (or the ratio of liposome to DNA) would not change the fact that the DNA and peptide are already interacting (ie, forming a complex) prior to liposome addition, so these experiments would not test the hypothesis. (Choice B) Excluding peptide from the assay to establish baseline levels of DNA delivery is an important control but does not test the importance of the order in which DNA must interact with peptides and liposomes. Educational objective: The order of interactions between biological molecules can affect the outcome of an experiment. This effect can be monitored by changing the order in which each component is added to an experimental system.

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