PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing

• Published in: International journal of molecular sciences Vol. 22; no. 18; p. 10025
• Main Authors: Aubets, Eva, Chillon, Miguel, Ciudad, Carlos J, Noé, Véronique
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 16.09.2021; MDPI
• Subjects: Acids; Adenoviridae - genetics; Apoptosis; Apoptosis - genetics; Apoptosis - physiology; Cancer therapies; Cell Survival - genetics; Cell Survival - physiology; Cell viability; delivery; Efficiency; Expression vectors; Gene expression; Gene silencing; Gene Silencing - physiology; gene targeting; Gene therapy; Genomes; HeLa Cells; Humans; Medical prognosis; mRNA; oligonucleotide; Oligonucleotides - genetics; Oligonucleotides - metabolism; PC-3 Cells; PPRH; Proteins; RNA-mediated interference; Survival; Survivin; Survivin - genetics; Survivin - metabolism; Transfection; Vectors (Biology); cancer therapy; delivery; survivin; PPRH; adenovirus; gene silencing; gene targeting; viral vectors; oligonucleotide
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms221810025

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 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 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.