43797026d410baea5cc0ca2d6c44c82b pmc_34576188.pdf 7d35b2c685ff6e670761160c96b7eccc99f7a226 pmc_34576188.pdf 82a16ac1844964a9c3324530cf975db20d763acd4fdfc2033750cfa985070725 pmc_34576188.pdf Title: PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing Subject: PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs. Keywords: PPRH; oligonucleotide; survivin; gene targeting; gene silencing; delivery; viral vectors; adenovirus; cancer therapy Author: Eva Aubets, Miguel Chillon, Carlos J. Ciudad and Véronique Noé Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Sep 18 09:09:23 2021 CEST ModDate: Sat Sep 18 09:13:31 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3378957 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WFLYQS+URWPalladioL-Ital Type 1 Custom yes yes no 10 0 TZNGJQ+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 CVDYYI+URWPalladioL-Roma Type 1 Custom yes yes no 19 0 BTFGED+VnURWPalladioL-Bold Type 1 Custom yes yes no 23 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 32 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 35 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 57 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 61 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 65 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 86 0 MDJLZU+EURM10 Type 1 Builtin yes yes no 96 0 OPZDHV+VnURWPalladioL Type 1 Custom yes yes no 130 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 138 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-11-23 05:27:32 CET RepresentationInformation: pmc_34576188.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:05:46 CEST Size: 3378957 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 478 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: RNA-PPRH Binding Analyses Destination: subsection.2.1 Item: Title: Effect of a Synthetic RNA-PPRH Targeting Survivin on Viability and Apoptosis Destination: subsection.2.2 Item: Title: Effect on the Levels of Survivin mRNA and Protein upon RNA-PPRH Transfection Destination: subsection.2.3 Item: Title: Cell Viability Assays with AdV Vectors Destination: subsection.2.4 Item: Title: Effect of AdV-PPRH Infection on Survivin mRNA and Protein Levels Destination: subsection.2.5 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Culture Destination: subsection.4.1 Item: Title: PPRHs Design Destination: subsection.4.2 Item: Title: RNA-PPRH Binding Analyses Destination: subsection.4.3 Item: Title: Plasmid Vector Destination: subsection.4.4 Item: Title: Viral Vector Production Destination: subsection.4.5 Item: Title: Transfection of PPRHs Destination: subsection.4.6 Item: Title: Transfection of Vector Destination: subsection.4.7 Item: Title: Transduction of Human Cells Destination: subsection.4.8 Item: Title: Cell Viability Assays Destination: subsection.4.9 Item: Title: Annexin V Apoptosis Detection Kit FITC Destination: subsection.4.10 Item: Title: RNA Extraction Destination: subsection.4.11 Item: Title: Reverse Transcription Destination: subsection.4.12 Item: Title: Reverse Transcription Destination: subsection.4.13 Item: Title: Western Blot Analyses for Survivin Detection Destination: subsection.4.14 Item: Title: Statistical Analyses Destination: subsection.4.15 Item: Title: References Destination: subsection.4.15 Info: Title: PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing Author: Eva Aubets, Miguel Chillon, Carlos J. Ciudad and Véronique Noé Subject: PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs. Keywords: PPRH; oligonucleotide; survivin; gene targeting; gene silencing; delivery; viral vectors; adenovirus; cancer therapy Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Sep 18 09:09:23 CEST 2021 ModDate: Sat Sep 18 09:13:31 CEST 2021 ID: 0x8d04fd0f1e8a49d0afae43f2e8676f5d, 0x8d04fd0f1e8a49d0afae43f2e8676f5d Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: MDJLZU+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: MDJLZU+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: OPZDHV+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: OPZDHV+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CVDYYI+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: CVDYYI+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true 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