Polypurine reverse Hoogsteen hairpins as a gene therapy tool against survivin in human prostate cancer PC3 cells in vitro and in vivo

Rodrı̀guez, Laura; Villalobos, Xenia; Dakhel, Sheila; Padilla, Laura; Hervás, Rubén; Hernández, José Luís; Ciudad, Carlos J.; Noé, Verónique

doi:10.1016/j.bcp.2013.09.013

Cited by 33 publications

(57 citation statements)

References 38 publications

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“…They can be easily synthesized since they do not bear chemical modifications, which is an advantage in terms of toxicity (Rodriguez et al, 2013) and economy. At the same time, due to their hairpin structure, PPRHs are known to be highly resistant to endogenous nucleases (Villalobos et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…We previously described the use of PPRHs as a silencing tool in both in vitro and in vivo approaches (de Almagro et al, 2009;Almagro et al, 2011;Rodriguez et al, 2013), by decreasing the expression of different targets. In this work we present evidence that PPRHs can also be used as a tool for gene repair.…”

Section: Discussionmentioning

confidence: 99%

“…We had previously described the design and usage of template-PPRHs that bind to the template DNA strand (de Almagro et al, 2009), and coding-PPRHs binding to the coding DNA strand (de Almagro et al, 2011), as a tool to decrease gene expression (Rodriguez et al, 2013) that shows high stability and low immunogenicity properties without the need for chemical modifications (Villalobos et al, 2014). In this work we explored a new application for PPRHs: the repair of point mutations, an ambitious gene therapy strategy.…”

Section: Introductionmentioning

confidence: 99%

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Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

Solé

Villalobos²,

Ciudad³

et al. 2014

Human Gene Therapy Methods

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Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with antiparallel orientation by Watson-Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

Solé

Villalobos²,

Ciudad³

et al. 2014

Human Gene Therapy Methods

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“…AR consensus double-stranded oligonucleotide 5′-CTA GAA GTC TGG TAC AGG GTG TTC TTT TTG CA-3′ (binding site in bold) was obtained from Santa Cruz Biotechnology, Heidelberg, Germany (sc-2551). One hundred nanograms of the AR consensus sequence was 5′-end-labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [γ-32 P]ATP (3000 Ci mmol −1 , Perkin Elmer, Madrid, Spain) as described by Rodríguez et al (2013). 46 The radiolabeled probe (20 000 cpm) was incubated in a 20 μL reaction mixture also containing 1 μg of Herring sperm DNA (Invitrogen, Barcelona, Spain) as an unspecific competitor, 2 μg of nuclear extract protein, 5% glycerol, 4 mM MgCI 2 , 60 mM KCl and 25 mM Tris-HCl, pH 8.0 (AppliChem, Barcelona, Spain).…”

Section: Electrophoretic Mobility Shift Assaymentioning

confidence: 99%

“…One hundred nanograms of the AR consensus sequence was 5′-end-labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [γ-32 P]ATP (3000 Ci mmol −1 , Perkin Elmer, Madrid, Spain) as described by Rodríguez et al (2013). 46 The radiolabeled probe (20 000 cpm) was incubated in a 20 μL reaction mixture also containing 1 μg of Herring sperm DNA (Invitrogen, Barcelona, Spain) as an unspecific competitor, 2 μg of nuclear extract protein, 5% glycerol, 4 mM MgCI 2 , 60 mM KCl and 25 mM Tris-HCl, pH 8.0 (AppliChem, Barcelona, Spain). Samples were resolved by gel electrophoresis (5% polyacrylamide, 5% glycerol, 1 mM EDTA and 45 mM Trisborate, pH 8.0; AppliChem, Barcelona, Spain).…”

Section: Electrophoretic Mobility Shift Assaymentioning

confidence: 99%

Walnut polyphenol metabolites, urolithins A and B, inhibit the expression of the prostate-specific antigen and the androgen receptor in prostate cancer cells

et al. 2014

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Walnuts have been gathering attention for their health-promoting properties. They are rich in polyphenols, mainly ellagitannins (ETs) that after consumption are hydrolyzed to release ellagic acid (EA). EA is further metabolized by microbiota to form urolithins, such as A and B, which are absorbed. ETs, EA and urolithins have shown to slow the proliferation and growth of different types of cancer cells but the mechanisms remain unclear. We investigate the role of urolithins in the regulatory mechanisms in prostate cancer, specifically those related to the androgen receptor (AR), which have been linked to the development of this type of cancer. In our study, urolithins down-regulated the mRNA and protein levels of both prostate specific antigen (PSA) and AR in LNCaP cells. The luciferase assay performed with a construct containing three androgen response elements (AREs) showed that urolithins inhibit AR-mediated PSA expression at the transcriptional level. Electrophoretic mobility shift assays revealed that urolithins decreased AR binding to its consensus response element. Additionally, urolithins induced apoptosis in LNCaP cells, and this effect correlated with a decrease in Bcl-2 protein levels. In summary, urolithins attenuate the function of the AR by repressing its expression, causing a down-regulation of PSA levels and inducing apoptosis. Our results suggest that a diet rich in ET-containing foods, such as walnuts, could contribute to the prevention of prostate cancer.

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Parallel Clamps and Polypurine Hairpins (PPRH) for Gene Silencing and Triplex‐Affinity Capture: Design, Synthesis, and Use

Aviñó

Eritja

Ciudad

et al. 2019

CP Nucleic Acid Chemistry

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Nucleic acid triplexes are formed when a DNA or RNA oligonucleotide binds to a polypurine‐polypyrimidine‐rich sequence. Triplexes have wide therapeutic applications such as gene silencing or site‐specific mutagenesis. In addition, protocols based on triplex‐affinity capture have been used for detecting nucleic acids in biosensing platforms. In this article, the design, synthesis, and use of parallel clamps and polypurine‐reversed hairpins (PPRH) to bind to target polypyrimidine targets are described. The combination of the polypurine Watson‐Crick strand with the triplex‐forming strand in a single molecule produces highly stable triplexes allowing targeting of single‐ and double‐stranded nucleic acid sequences. On the other hand, PPRHs are easily prepared and work at nanomolar range, like siRNAs, and at a lower concentration than that needed for antisense ODNs or TFOs. However, the stability of PPRHs is higher than that of siRNAs. In addition, PPRHs circumvent off‐target effects and are non‐immunogenic. © 2019 by John Wiley & Sons, Inc.

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Polypurine reverse Hoogsteen hairpins as a gene therapy tool against survivin in human prostate cancer PC3 cells in vitro and in vivo

Cited by 33 publications

References 38 publications

Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

Walnut polyphenol metabolites, urolithins A and B, inhibit the expression of the prostate-specific antigen and the androgen receptor in prostate cancer cells

Parallel Clamps and Polypurine Hairpins (PPRH) for Gene Silencing and Triplex‐Affinity Capture: Design, Synthesis, and Use

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