Gene Silencing Mediated by siRNA-binding Fusion Proteins Is Attenuated by Double-stranded RNA-binding Domain Structure
Delivery of small interfering RNA (siRNA) targeted to specific cell types is a significant challenge for the development of RNA interference-based therapeutics. Recently, PTD-DRBD, a double-stranded RNA binding domain (DRBD) fused to the TAT protein transduction domain (PTD), was shown to be effecti...
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| Format: | Article |
| Language: | English |
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Elsevier
2012-01-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253116301093 |
| _version_ | 1828737889983266816 |
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| author | James C Geoghegan Brian L Gilmore Beverly L Davidson |
| author_facet | James C Geoghegan Brian L Gilmore Beverly L Davidson |
| author_sort | James C Geoghegan |
| collection | DOAJ |
| description | Delivery of small interfering RNA (siRNA) targeted to specific cell types is a significant challenge for the development of RNA interference-based therapeutics. Recently, PTD-DRBD, a double-stranded RNA binding domain (DRBD) fused to the TAT protein transduction domain (PTD), was shown to be effective at delivering siRNA in a non-cell type-specific manner. Here, we evaluated the potential of DRBD as a general protein platform for targeted small interfering RNA (siRNA) delivery. We found that a single DRBD was insufficient to stably complex siRNA when fused to targeting peptides other than PTD, which facilitated nonspecific nucleic acid binding. In contrast to PTD-DRBD, fusion proteins containing two DRBDs (2× DRBD) yielded specific and stable siRNA binding. These proteins could mediate the cellular uptake of siRNA in vitro, though compared with PTD-DRBD gene silencing was attenuated by endosomal entrapment. Our findings suggest that unlike a single DRBD, 2× DRBD inhibits siRNA escape into the cytoplasm and/or induces an internalization pathway distinct from that of PTD-DRBD. Collectively, these data indicate that while 2× DRBD retains siRNA-binding activity when fused to different cell surface-interacting peptides, the utility of 2× DRBD for cell-specific RNA interference is limited without further protein engineering to enhance the bioavailability of the delivered siRNAs. |
| first_indexed | 2024-04-12T23:52:01Z |
| format | Article |
| id | doaj.art-fc55601d30104a9fa8c1b0a8f799ea48 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-12T23:52:01Z |
| publishDate | 2012-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-fc55601d30104a9fa8c1b0a8f799ea482022-12-22T03:11:40ZengElsevierMolecular Therapy: Nucleic Acids2162-25312012-01-011C10.1038/mtna.2012.43Gene Silencing Mediated by siRNA-binding Fusion Proteins Is Attenuated by Double-stranded RNA-binding Domain StructureJames C Geoghegan0Brian L Gilmore1Beverly L Davidson2Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USADepartment of Internal Medicine, University of Iowa, Iowa City, Iowa, USADepartment of Internal Medicine, University of Iowa, Iowa City, Iowa, USADelivery of small interfering RNA (siRNA) targeted to specific cell types is a significant challenge for the development of RNA interference-based therapeutics. Recently, PTD-DRBD, a double-stranded RNA binding domain (DRBD) fused to the TAT protein transduction domain (PTD), was shown to be effective at delivering siRNA in a non-cell type-specific manner. Here, we evaluated the potential of DRBD as a general protein platform for targeted small interfering RNA (siRNA) delivery. We found that a single DRBD was insufficient to stably complex siRNA when fused to targeting peptides other than PTD, which facilitated nonspecific nucleic acid binding. In contrast to PTD-DRBD, fusion proteins containing two DRBDs (2× DRBD) yielded specific and stable siRNA binding. These proteins could mediate the cellular uptake of siRNA in vitro, though compared with PTD-DRBD gene silencing was attenuated by endosomal entrapment. Our findings suggest that unlike a single DRBD, 2× DRBD inhibits siRNA escape into the cytoplasm and/or induces an internalization pathway distinct from that of PTD-DRBD. Collectively, these data indicate that while 2× DRBD retains siRNA-binding activity when fused to different cell surface-interacting peptides, the utility of 2× DRBD for cell-specific RNA interference is limited without further protein engineering to enhance the bioavailability of the delivered siRNAs.http://www.sciencedirect.com/science/article/pii/S2162253116301093DRBDprotein transduction domainRNA interferencesiRNA delivery |
| spellingShingle | James C Geoghegan Brian L Gilmore Beverly L Davidson Gene Silencing Mediated by siRNA-binding Fusion Proteins Is Attenuated by Double-stranded RNA-binding Domain Structure Molecular Therapy: Nucleic Acids DRBD protein transduction domain RNA interference siRNA delivery |
| title | Gene Silencing Mediated by siRNA-binding Fusion Proteins Is Attenuated by Double-stranded RNA-binding Domain Structure |
| title_full | Gene Silencing Mediated by siRNA-binding Fusion Proteins Is Attenuated by Double-stranded RNA-binding Domain Structure |
| title_fullStr | Gene Silencing Mediated by siRNA-binding Fusion Proteins Is Attenuated by Double-stranded RNA-binding Domain Structure |
| title_full_unstemmed | Gene Silencing Mediated by siRNA-binding Fusion Proteins Is Attenuated by Double-stranded RNA-binding Domain Structure |
| title_short | Gene Silencing Mediated by siRNA-binding Fusion Proteins Is Attenuated by Double-stranded RNA-binding Domain Structure |
| title_sort | gene silencing mediated by sirna binding fusion proteins is attenuated by double stranded rna binding domain structure |
| topic | DRBD protein transduction domain RNA interference siRNA delivery |
| url | http://www.sciencedirect.com/science/article/pii/S2162253116301093 |
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