Evaluation Of Fusion Peptides As Novel Gene Carriers Into Nicotiana Benthamiana And Arabidopsis Thaliana Leaves
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Date
2016-02
Authors
Lakshmanan, Manoj Kumar
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Universiti Sains Malaysia
Abstract
Peptide-based gene delivery involves introduction of foreign genes into animal and plant cells via functional peptides, containing cell penetrating domains and polycationic sequences. Cell penetrating domains are capable of being internalized into living cells, while polycationic sequences can electrostatically interact and condense DNA. In this study, three fusion peptides consisting of polycationic sequences and functional domains with cell penetrating ability were designed and evaluated as potential gene carriers for plant cells. The fusion peptides, consisting of nona-arginine (R9) and histidine-lysine (KH)9 polycationic sequences as well as Bp100 (KKLFKKILKYL) and Tat2 (RKKRRQRRRRKKRRQRRR) cell penetrating domains respectively were mixed with pDNA encoding Renilla luciferase (RLuc) to form pDNA-peptide complexes at various N/P ratios. Here, N/P ratio refers to the ratio of number of amines from peptides per number of phosphates from pDNA. The complexes were characterized in terms of size, surface charges (zeta potential), stability and morphology. The complexes prepared at various N/P ratios (0.1, 0.5, 1, 2, 5, 10 and 20) in deionized water were infiltrated at the abaxial section of 3-week old Nicotiana benthamiana and Arabidopsis thaliana leaves and quantified for RLuc gene expression using RLuc assay at various time points up to 144 hours. Complexes of (KH)9-Bp100 prepared at N/P ratio 0.5 demonstrated globular shapes with hydrodynamic diameters between 300-400 nm and negatively charged surface. This complexes also showed the highest transfection efficiencies at 12 hours after infiltration for all the fusion peptides compared to the other N/P ratios. Based on the observation obtained in this first section, the pDNA-peptide complexes were further evaluated in different buffers and pH. For this, complexes were first prepared at various N/P ratios (0.1, 0.5, 1, 2 and 5) and characterized in terms of size, surface charge and morphology. Transfection efficiency studies performed on N. benthamiana leaves suggested that pDNA-peptide complexes at N/P ratio 0.5 still showed the highest levels of efficiencies in buffer solution (30 mM, PBS, pH 7.4) along with the results obtained in deionized water. In the third stage, a linear double stranded DNA (dsDNA) encoding RLuc genes were synthesized using PCR methods with the pDNA encoding RLuc as the template and used to prepare dsDNA-peptide complexes in both deionized water and buffers at various N/P ratios. The dsDNA-peptide complexes at N/P ratio 1 gave highest transfection efficiencies in both deionized water and buffer conditions (30 mM, PBS, pH 7.4).
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