RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure
Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passeng...
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2014-01-01
|
| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253116302803 |
| _version_ | 1818473768507408384 |
|---|---|
| author | Simon J Allison Jo Milner |
| author_facet | Simon J Allison Jo Milner |
| author_sort | Simon J Allison |
| collection | DOAJ |
| description | Selective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3′ end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3′-DNA/ds siRNA and includes a 3′-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3′-DNA/ds siRNA, (II) passenger-3′-DNA/ds siRNA, (III) guide-3′-DNA/ss siRNA, and (IV) passenger-3′-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I–III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3′-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3′-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3′-DNA/siRNA constructs are discussed. |
| first_indexed | 2024-04-14T04:27:50Z |
| format | Article |
| id | doaj.art-df0616dfb15448c78db65efedd9e79e8 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-14T04:27:50Z |
| publishDate | 2014-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-df0616dfb15448c78db65efedd9e79e82022-12-22T02:12:15ZengElsevierMolecular Therapy: Nucleic Acids2162-25312014-01-013C10.1038/mtna.2013.68RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin StructureSimon J Allison0Jo Milner1Department of Biology, University of York, York, UKDepartment of Biology, University of York, York, UKSelective gene silencing by RNA interference (RNAi) involves double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs. siRNA is recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). RISC cleaves passenger RNA, exposing the guide RNA for base-pairing with its homologous mRNA target. Remarkably, the 3′ end of passenger RNA can accommodate a DNA extension of 19-nucleotides without loss of RNAi function. This construct is termed passenger-3′-DNA/ds siRNA and includes a 3′-nuclease-resistant mini-hairpin structure. To test this novel modification further, we have now compared the following constructs: (I) guide-3′-DNA/ds siRNA, (II) passenger-3′-DNA/ds siRNA, (III) guide-3′-DNA/ss siRNA, and (IV) passenger-3′-DNA/ss siRNA. The RNAi target was SIRT1, a cancer-specific survival factor. Constructs I–III each induced selective knock-down of SIRT1 mRNA and protein in both noncancer and cancer cells, accompanied by apoptotic cell death in the cancer cells. Construct IV, which lacks the SIRT1 guide strand, had no effect. Importantly, the 3′-DNA mini-hairpin conferred nuclease resistance to constructs I and II. Resistance required the double-stranded RNA structure since single-stranded guide-3′-DNA/ss siRNA (construct III) was susceptible to serum nucleases with associated loss of RNAi activity. The potential applications of 3′-DNA/siRNA constructs are discussed.http://www.sciencedirect.com/science/article/pii/S2162253116302803Ago2C3POnuclease-resistant DNA mini-hairpinRNAiSIRT1therapy3′-DNA/siRNA constructs |
| spellingShingle | Simon J Allison Jo Milner RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure Molecular Therapy: Nucleic Acids Ago2 C3PO nuclease-resistant DNA mini-hairpin RNAi SIRT1 therapy 3′-DNA/siRNA constructs |
| title | RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure |
| title_full | RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure |
| title_fullStr | RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure |
| title_full_unstemmed | RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure |
| title_short | RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3′ Nuclease-resistant Mini-hairpin Structure |
| title_sort | rna interference by single and double stranded sirna with a dna extension containing a 3 nuclease resistant mini hairpin structure |
| topic | Ago2 C3PO nuclease-resistant DNA mini-hairpin RNAi SIRT1 therapy 3′-DNA/siRNA constructs |
| url | http://www.sciencedirect.com/science/article/pii/S2162253116302803 |
| work_keys_str_mv | AT simonjallison rnainterferencebysingleanddoublestrandedsirnawithadnaextensioncontaininga3nucleaseresistantminihairpinstructure AT jomilner rnainterferencebysingleanddoublestrandedsirnawithadnaextensioncontaininga3nucleaseresistantminihairpinstructure |