Part:BBa_K3224003
BP100-(KH)₉
Cell-Penetrating Peptides (CPP) are short peptides that can penetrate the cell membrane and deliver a wide range of cargoes into the cell. This part is the coding sequence of our chimeric CPP formed with BP100 and the polycationic peptide (KH)9. It is proven to deliver proteins and nucleic acids into the plant and prokaryotic cells. The polycationic peptide is an efficient nucleic acid carrier and has the ability to promote buffering effect on the pre-lysosomal vesicle. The cargo being negatively charged interacts with the polycationic end of the peptide through ionic interactions, whereas the BP100 region mediates cellular entry through carpet-mechanism.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
CHARACTERIZATION
To determine the cytotoxicity of the peptide produced by BBa_K3224003 (Cell Penetrating Peptide - BP100(KH)9) in HeLa (immortalized human cervical cancer) and C2C12 (immortalized mouse myoblast) cell.
The cells were treated with different concentrations of the CPP encoded by the biobrick (4,8,16,32,64,120 μM/ml) of BP-100-(KH)9 and were incubated for different time intervals 24 h and 48 h.
MTT ASSAY OF CPP IN HELA CELLS (24 HRS)
FIGURE 1: MTT Assay of CPP in HeLa Cells (24 h)
When HeLa cells were treated with lower concentrations of CPP (4-16 µM) for 48 hours, ~15% cytotoxicity was observed. Higher concentrations of CPP (32-120 µM) showed ~25% cytotoxicity on the cells.
MTT ASSAY OF CPP IN HELA CELLS (48 HRS)
FIGURE 2: MTT Assay of CPP in HeLa cells (48 h)
When HeLa cells were treated with CPP for a time period of 48 hours, the cytotoxicity levels lie below 50% for lower concentrations of the peptide (4 and 8µM). For the concentrations from 16 to 120µM the cytotoxicity shoots up to ~100%.
MTT ASSAY OF CPP IN C2C12 CELLS (24 HRS)
FIGURE 3: MTT Assay of CPP in C2C12 Cells (24 h)
The action of lower concentrations of CPP (4 and 8µM) on C2C12 cells showed ~35% cytotoxicity. At higher concentrations of CPP (16 to120µM) ~135% cytotoxicity was observed.
MTT ASSAY OF CPP IN C2C12 CELLS (48 HRS)
FIGURE 4: MTT Assay of CPP in C2C12 Cells (48 h)
When C2C12 cell lines were treated with lower concentrations of CPP (4-32µM) for 48 hours, CPP showed ~100% cytotoxicity. At higher concentrations (64 and 120µM) the cytotoxicity goes beyond 150%.
OPTIMIZATION OF N:P RATIO:
The Cell Penetrating Peptide BP100-(KH)9 (BBa_K3224003) was synthesized in-vitro and purified by High-Performance Liquid Chromatography (HPLC). The in-vitro synthesized shRNA-like siRNA with a fluorophore and a quencher (BBa-K3224002) was mixed at 100 µM concentration, with varying concentrations of the chimeric CPP BP100- (KH)9 to make up different N/P ratios: 0.5, 1 and 2. Also, only siRNA without the CPP was added to the cells as delivery control. The fluorescence emitted was measured in Arbitrary Fluorescence Units (AFU). The other nucleotide construct, survivin shRNA (BBa_K3224001), was not used for this experiment as it is devoid of a fluorophore.
The intensity of the fluorescence emitted is proportional to the amount of cargo successfully delivered into the cells, and hence an indicator of the optimum N/P ratio in order to achieve improved transfection efficiency. From the graph, we observe that the N/P ratio of 0.5 gives higher intensity of fluorescence which can be attributed to the presence of higher amount of CPP for a given amount of siRNA to be transfected. Thus the efficient delivery of the nucleic acid has taken place by the CPP.
As the amount of protein used to make up the N:P ratios of 1.0 and 2.0 for the fixed concentration of nucleotide (100 µL) decreases, lesser number of nucleotides get delivered into the cells, which in turn accounts for the decrease in fluorescence emitted from the cells in these two experimental conditions. In addition to optimizing the N:P ratio, the current experiment also confirms that the two submitted parts work: CPP in terms of delivering the cargo, and shRNA-like siRNA in terms of emitting fluorescence.
Figure 5: Optimization of the N/P ratio of CPP-siRNA complexes
Contribution: New data added by SZU-China 2023
Biology
Cell-penetrating peptides (CPPs) is a short chain amino acid composed of about 30 amino acids, including basic amino acids and R groups, which can be used to deliver biological molecules such as siRNA, pDNA, plasmid and protein, and has been relatively mature in the medical field. Engineered CPP and shRNA molecules interact via positively charged amino acid residues such as arginine and lysine with the phosphate skeleton of negatively charged nucleic acids to form submicron peptide-nucleic acid complexes. This non-covalent binding method has stability and reversibility, thus realizing the transfer and function of biomolecules.
KH9-BP100 is a CPP that fuses the highly potent cell-penetrating peptide BP100 with the biomolecular binding domain KH9 to improve the delivery efficiency of biomolecules into plant cells. KH9-BP100 shows a spherical shape under the Atomic force microscope (AFM) and can wrap shRNA molecules to form spherical complexes. The combination of KH9-BP100 and shRNA is simple, just mix the two substances in a certain proportion, and let them stand at room temperature for a period of time to obtain the complex of CPP-shRNA. By spraying CPP-shRNAs on tomatoes infected by B. cinerea, shRNAs can enter plant cells more efficiently under the effect of CPP. Then, siRNAs are formed after processing of Dicer/ Dicer-like proteins in cells, which will be next delivered to B. cinerea, to achieve the effect of silencing the key genes of B. cinerea infection and its own survival genes.
Characterization
CPP-shRNA under SEM
However, the instability of shRNA in the field environment hinders the optimal performance of our product. Understanding our expectations, our PI suggested that we could try using cell-penetrating peptides (CPP) in combination with shRNA for spray application, and provided us with KH9-BP100 as our CPP material. KH9-BP100 is a carrier peptide-based gene delivery system that enhances the endocytic uptake and cytoplasmic transfer of shRNA in plants, allowing for more efficient transfection of plant callus cells with shRNA. To understand the morphology of the shRNA and KH9-BP100 complex, we used scanning electron microscopy (SEM) to observe the morphology of shRNA, KH9-BP100, and shRNA+KH9-BP100 separately. As shown in the figure, we observed that CPP-shRNA complex form spherical aggregates under electron microscopy. These small spherical aggregates further tend to aggregate with each other. We speculate that this stacking aggregation is due to electrostatic forces.
Distribution of disease spots
We designed shRNA to target and silence genes necessary for the survival of B. cinerea and virulence genes of infected tomatoes. Therefore, we wanted to test whether spraying shRNA can really reduce the attack of B. cinerea on tomato fruits. We used a black marker to draw a circle with a diameter of about 3 mm on the surface of the tomato fruit, and poked five small holes within the circle with a sterilized thin needle.
For the naked shRNA treatment, we added 10 μL solution containing 10 μg shRNA in and around the circle of the fruit surface. After the liquid dried, we drilled holes on the edge of the B. cinerea plate with a 10 μL transparent suction head, and then covered the surface of the fruit in the dotted line area with the mycelium side of the cake. In the control group, we selected non-specific shRNA GFP. The treated tomato fruits were placed in a humid environment at 21 ℃. After three days, ImageJ software was used to conduct a quantitative analysis of the lesion area, which was determined by the area covered by mycelia. Error bars represent standard deviations (SD) obtained from 11-15 biological replicates, the data are F-tested and T-tested, the level of significant difference is passed by a single-tail test, and shown above the bar chart (ns P > 0.05；* P < 0.05；** P < 0.01；*** P < 0.001). For the treatment coated with transmembrane peptide (CPP), the treatment was consistent with the naked shRNA treatment, except that the solution of dripping per sample was changed to 12 μL containing 10 μg shRNA and 8.2 μL 1mg/mL CPP (Figure 2).
At the phenotypic level, the shRNA(DCL2)-1 treatment was no significant difference compared with the control group. When combined with CPP, the relative plaque area treated by shRNA(DCL2)-1 was reduced by 21.9% compared with control, proving that CPP can help shRNA more effective.
Detection of inhibition effect by qRT-PCR
On the third day of the experiment, after sampling the lesions of tomato fruits, the sample RNA was extracted, reverse-transcribed, and qRT-PCR was performed to detect the inhibition effect of shRNA on mycelium target mRNA in infected fruits (Figure 3).
From the results of molecular experiments, the silencing rates of the experiment that spraying the naked shRNA(DCL2)-1 is 46.8%, after binding with CPP, the silencing rates could reach 58.5%. This result have showed that CPP was really poly a good role in helping shRNA to silencing target mRNA.
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