Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide
Abstract Background Although protein-based methods using cell-penetrating peptides such as TAT have been expected to provide an alternative approach to siRNA delivery, the low efficiency of endosomal escape of siRNA/protein complexes taken up into cells by endocytosis remains a problem. Here, to ove...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-10-01
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| Series: | Journal of Nanobiotechnology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12951-022-01667-4 |
| _version_ | 1797991657602285568 |
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| author | Momoko Nakamura Kei Fujiwara Nobuhide Doi |
| author_facet | Momoko Nakamura Kei Fujiwara Nobuhide Doi |
| author_sort | Momoko Nakamura |
| collection | DOAJ |
| description | Abstract Background Although protein-based methods using cell-penetrating peptides such as TAT have been expected to provide an alternative approach to siRNA delivery, the low efficiency of endosomal escape of siRNA/protein complexes taken up into cells by endocytosis remains a problem. Here, to overcome this problem, we adopted the membrane penetration-enhancing peptide S19 from human syncytin 1 previously identified in our laboratory. Results We prepared fusion proteins in which the S19 and TAT peptides were fused to the viral RNA-binding domains (RBDs) as carrier proteins, added the RBD-S19-TAT/siRNA complex to human cultured cells, and investigated the cytoplasmic delivery of the complex and the knockdown efficiency of target genes. We found that the intracellular uptake of the RBD-S19-TAT/siRNA complex was increased compared to that of the RBD-TAT/siRNA complex, and the expression level of the target mRNA was decreased. Because siRNA must dissociate from RBD and bind to Argonaute 2 (Ago2) to form the RNA-induced silencing complex (RISC) after the protein/siRNA complex is delivered into the cytoplasm, a dilemma arises: stronger binding between RBD and siRNA increases intracellular uptake but makes RISC formation more difficult. Thus, we next prepared fusion proteins in which the S19 and TAT peptides were fused with Ago2 instead of RBD and found that the efficiencies of siRNA delivery and knockdown obtained using TAT-S19-Ago2 were higher than those using TAT-Ago2. In addition, we found that the smallest RISC delivery induced faster knockdown than traditional siRNA lipofection, probably due to the decreased time required for RISC formation in the cytoplasm. Conclusion These results indicated that S19 and TAT-fused siRNA-binding proteins, especially Ago2, should be useful for the rapid and efficient delivery of siRNA without the addition of any endosome-disrupting agent. |
| first_indexed | 2024-04-11T08:55:43Z |
| format | Article |
| id | doaj.art-f758b0545c934816a1e626ccdbe4ec35 |
| institution | Directory Open Access Journal |
| issn | 1477-3155 |
| language | English |
| last_indexed | 2024-04-11T08:55:43Z |
| publishDate | 2022-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Nanobiotechnology |
| spelling | doaj.art-f758b0545c934816a1e626ccdbe4ec352022-12-22T04:33:19ZengBMCJournal of Nanobiotechnology1477-31552022-10-0120111110.1186/s12951-022-01667-4Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptideMomoko Nakamura0Kei Fujiwara1Nobuhide Doi2Department of Biosciences and Informatics, Keio UniversityDepartment of Biosciences and Informatics, Keio UniversityDepartment of Biosciences and Informatics, Keio UniversityAbstract Background Although protein-based methods using cell-penetrating peptides such as TAT have been expected to provide an alternative approach to siRNA delivery, the low efficiency of endosomal escape of siRNA/protein complexes taken up into cells by endocytosis remains a problem. Here, to overcome this problem, we adopted the membrane penetration-enhancing peptide S19 from human syncytin 1 previously identified in our laboratory. Results We prepared fusion proteins in which the S19 and TAT peptides were fused to the viral RNA-binding domains (RBDs) as carrier proteins, added the RBD-S19-TAT/siRNA complex to human cultured cells, and investigated the cytoplasmic delivery of the complex and the knockdown efficiency of target genes. We found that the intracellular uptake of the RBD-S19-TAT/siRNA complex was increased compared to that of the RBD-TAT/siRNA complex, and the expression level of the target mRNA was decreased. Because siRNA must dissociate from RBD and bind to Argonaute 2 (Ago2) to form the RNA-induced silencing complex (RISC) after the protein/siRNA complex is delivered into the cytoplasm, a dilemma arises: stronger binding between RBD and siRNA increases intracellular uptake but makes RISC formation more difficult. Thus, we next prepared fusion proteins in which the S19 and TAT peptides were fused with Ago2 instead of RBD and found that the efficiencies of siRNA delivery and knockdown obtained using TAT-S19-Ago2 were higher than those using TAT-Ago2. In addition, we found that the smallest RISC delivery induced faster knockdown than traditional siRNA lipofection, probably due to the decreased time required for RISC formation in the cytoplasm. Conclusion These results indicated that S19 and TAT-fused siRNA-binding proteins, especially Ago2, should be useful for the rapid and efficient delivery of siRNA without the addition of any endosome-disrupting agent.https://doi.org/10.1186/s12951-022-01667-4Argonaute 2Cell-penetrating peptideEndosomal escapeFusogenic peptideGene knockdown |
| spellingShingle | Momoko Nakamura Kei Fujiwara Nobuhide Doi Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide Journal of Nanobiotechnology Argonaute 2 Cell-penetrating peptide Endosomal escape Fusogenic peptide Gene knockdown |
| title | Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide |
| title_full | Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide |
| title_fullStr | Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide |
| title_full_unstemmed | Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide |
| title_short | Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide |
| title_sort | cytoplasmic delivery of sirna using human derived membrane penetration enhancing peptide |
| topic | Argonaute 2 Cell-penetrating peptide Endosomal escape Fusogenic peptide Gene knockdown |
| url | https://doi.org/10.1186/s12951-022-01667-4 |
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